{"aops":[{"id":3,"title":"Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits","short_name":"Mitochondrial dysfunction and Neurotoxicity","corresponding_author_id":27,"abstract":"\u003cp\u003eThis Adverse outcome Pathway (AOP) describes the linkage between inhibition of complex I (CI) of the mitochondrial respiratory chain and motor deficit as in parkinsonian disorders. Binding of an inhibitor to complex I has been defined as the molecular initiating event (MIE) that triggers mitochondrial dysfunction, impaired proteostasis, which then cause degeneration of dopaminergic (DA) neurons of the nigro-striatal pathway. Neuroinflammation is triggered early in the neurodegenerative process and exacerbates it significantly. These causatively linked cellular key events result in motor deficit symptoms, typical for parkinsonian disorders, including Parkinson\u0026#39;s disease (PD), described in this AOP as an Adverse Outcome (AO). Since the release of dopamine in the striatum by DA neurons of the Substantia Nigra pars compacta (SNpc) is essential for motor control, the key events refer to these two brain structures. The weight-of-evidence supporting the relationship between the described key events is based mainly on effects observed after an exposure to the chemicals rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), i.e. two well-known inhibitors of complex I. Data from experiments with these two chemicals reveal a significant concordance in the dose-response relationships between the MIE and AO and within key events (KEs). Also essentiality of the described KEs for this AOP is strong since there is evidence from knock out animal models, engineered cells or replacement therapies that blocking, preventing or attenuating an upstream KE is mitigating the AO. Similarly, there is proved experimental support for the key event relationships (KERs) as multiple studies performed with modulating factors that attenuate (particularly with antioxidants) or augment (e.g. overexpression of viral-mutated \u0026alpha;-synuclein) a KE up show that such interference leads to an increase of KE down or the AO. Information from in vitro and in vivo experiments is complemented by human studies in brain tissues from individuals with sporadic Parkinson\u0026#39;s disease (Keeney et al., 2006) to support the pathways of toxicity proposed in this AOP.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:15.000-05:00","updated_at":"2025-10-04T02:11:56.000-04:00","status_id":1,"authors":"\u003cp\u003eAnna Bal-Price, European Commission, Joint Research Centre (JRC), Ispra, Italy\u003c/p\u003e\r\n\r\n\u003cp\u003eMarcel Leist, University of Konstanz, Konstanz, Germany\u003c/p\u003e\r\n\r\n\u003cp\u003eStefan Schildknecht, University of Konstanz, Konstanz, Germany\u003c/p\u003e\r\n\r\n\u003cp\u003eFlorianne Tschudi-Monnet, University of Lausanne and SCAHT, Lausanne, Switzerland\u003c/p\u003e\r\n\r\n\u003cp\u003eAlicia Paini, European Commission, Joint Research Centre (JRC), Ispra, Italy\u003c/p\u003e\r\n\r\n\u003cp\u003eAndrea Terron, European Food Safety Authority, Parma Italy (corresponding author: Andrea.TERRON@efsa.europa.eu)\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e- Revision of AOP3 (Project: \u003ca href=\"https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms\" rel=\"noreferrer noopener\" target=\"_blank\"\u003eNP/EFSA/PREV/2024/02\u003c/a\u003e):\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eBarbara Viviani, Department of Pharmacological and Biomolecular Sciences, Universit\u0026agrave; degli Studi di Milano, Italy\u003c/p\u003e\r\n\r\n\u003cp\u003eJo Nyffeler,\u0026nbsp;Department Ecotoxicology | Chemicals in the Environment Research Section, Helmholtz Centre for Environmental Research-UFZ, Germany\u003c/p\u003e\r\n\r\n\u003cp\u003eCleo Tebby,\u0026nbsp;Ineris (Institut national de l\u0026rsquo;environnement industriel et des risques),\u0026nbsp;Verneuil-en-Halatte, France\u003c/p\u003e\r\n\r\n\u003cp\u003eStefan Schildknecht,\u0026nbsp;Hochschule Albstadt-Sigmaringen,\u0026nbsp;Sigmaringen, Germany\u003c/p\u003e\r\n\r\n\u003cp\u003eR\u0026eacute;my\u0026nbsp;Beaudouin,\u0026nbsp;Ineris (Institut national de l\u0026rsquo;environnement industriel et des risques),\u0026nbsp;Verneuil-en-Halatte, France\u003c/p\u003e\r\n\r\n\u003cp\u003eEmma De Fabiani,\u0026nbsp;Department of Pharmacological and Biomolecular Sciences, Universit\u0026agrave; degli Studi di Milano, Italy\u003c/p\u003e\r\n\r\n\u003cp\u003eGiacomo Grumelli,\u0026nbsp;Department of Pharmacological and Biomolecular Sciences, Universit\u0026agrave; degli Studi di Milano, Italy\u003c/p\u003e\r\n\r\n\u003cp\u003eMilad Mohammadi,\u0026nbsp;Department Ecotoxicology | Chemicals in the Environment Research Section, Helmholtz Centre for Environmental Research-UFZ, Germany\u003c/p\u003e\r\n\r\n\u003cp\u003eInformation specialists: Elena Bernardini, Flavia Rampichini,\u0026nbsp;Universit\u0026agrave; degli Studi di Milano, Italy\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003eThis proposed AOP is neither sex-dependent nor associated with certain life stage; however, aged animals may be more sensitive. The relevance of this AOP during the developmental period has not been investigated. In vivo testing has no species restriction. The mouse was the species most commonly used in the experimental models conducted with the chemical stressors; though experimental studies using alternative species have been also performed. (Johnson et al. 2015). However, animal models (rodents in particular) would have limitations as they are poorly representative of the long human life-time as well as of the human long-time exposure to the potential toxicants. Human cell-based models would likely have better predictivity for humans than animal cell models. In this case, toxicokinetics information from in-vivo studies would be essential to test the respective concentrations in-vitro on human cells.\u003c/p\u003e\r\n","key_event_essentiality":"\u003cp\u003eEssentiality of KEs for this AOP is strong. There is ample evidence from knock out animal models, engineered cells or replacement therapies that blocking, preventing or attenuating an upstream KE is mitigating the AO. In addition, there is experimental support for the KERs as multiple studies performed with modulating factors that attenuate (particularly with antioxidants) or augment (e.g. overexpression of viral-mutated \u0026alpha;-synuclein) a KE show that such interference leads to an increase of KE down or the AO.\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003e2 Support for Essentiality of KEs\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eDefining Question\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eAre downstream KEs and/or the AO prevented if an upstream KE is blocked\u0026nbsp;?\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eHigh (Strong)\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eModerate\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eLow(Weak)\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eDirect evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g. stop/reversibility studies, antagonism, knock out models, etc.)\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eIndirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eNo or contradictory experimental evidence of the essentiality of any of the KEs\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE1\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eInhibition of complex I\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: Inactivation of the NADH:Ubiquinone Oxidoreductase Core Subunit S7\u0026nbsp;(Ndufs 4 gene knockout mice) that produces CI deficiency causes encephalomyopathy, including ataxia and loss of motor skills (Kruse et al., 2008). NDI1-transducted SK-N-MC cells expressing the rotenone-insensitive single subunit NADH dehydrogenase of yeast (NDI1) that acts as a replacement for the entire CI in mammalian cells were completely resistant to\u0026nbsp;100 nM rotenone, 100 nM fenpyroximate or 1 uM tebufenpyrad-mediated cell death (at 48 hrs of exposure) indicating that cI inhibitors\u0026nbsp;\u0026ndash; induced toxicity requires their\u0026nbsp;biding of CI (Sherer et al., 2003). In all rotenone models, mitochondria CI is inhibited at the dose that cause neurodegeneration (Betarbet et al 2000 and 2006).\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e- Revision of AOP3 (Project:\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e \u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003ca href=\"https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms\" style=\"color:#467886; text-decoration:underline\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eNP/EFSA/PREV/2024/02\u003c/span\u003e\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e:\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eThe mouse model, MCI-Park, by selectively deleting Ndufs2, an essential subunit of MCI, in dopaminergic neurons using intersectional genomics, reproduces several key features of progressive parkinsonism, including impaired dopamine release from striatal axons and deficits in associative learning. These mice initially displayed only mild fine motor deficits, while severe movements impairment responsive to levo-dopa emerged later in life, mirroring aspects of PD pathology. This progression coincided with the spread of dopaminergic signaling deficits from the striatum to the substantia nigra (Gonzalez-Rodriguez et al. 2020).\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e- Not endorsed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE2\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eMitochondrial dysfunction\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: Many studies showing that antioxidants protect the cells against cI inhibitors\u0026nbsp;induced oxidative stress are published (Chen et al. 2015; Lu et al., 2015; Saravanan et al., 2006; Chiu et al., 2015, Sherer et al.2003, Nataraj et al.2015, Wu et al. 1994; Tseng et al. 2014; Li et al. 2010; Kim-Han et al. 2011). This provides (indirect) evidence for essentiality of KE2, if production of reactive oxygen species (ROS) is assumed as direct consequence/sign of mitochondrial dysfunction. Additional evidence comes from experiments with overexpression or activation of antioxidative enzymes (e.g.SOD or ALDH2) , which also prevent rotenone and MPTP/MPP\u003csup\u003e+\u003c/sup\u003e induced neurotoxicity (Mudo et al. 2012; Ciu CC et al. 2015). Furthermore, promotion of mitochondrial fusion or blocking of mitochondrial fission prevents or attenuates rotenone and MPTP/MPP\u003csup\u003e+\u003c/sup\u003e induced neurotoxicity (Tieu K. et al. 2014).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE3 \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eImpaired proteostasis\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMODERATE\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: Indirect evidence for the role of disturbed alpha-synuclein proteostasis: Lacking of alpha-synuclein expression in mice prevented induction of behavioural symptoms, neuronal degeneration in the nigrostriatal pathway and loss of DA neurons after chronic treatment with MPTP/MPP\u003csup\u003e+\u003c/sup\u003e (Fornai et al. 2004; Dauer et al. 2002) . Injection of adeno/lenti-associated virus that expresses wild-type or mutant \u0026alpha;-synuclyn into rat, mice or non-human primate SN produced loss of dopaminergic neurons, but the effect is not easily reproduced in transgenic mice overexpressing alpha-synuclein (Kirk, 2002; Klein, 2002; Lo Bianco, 2002; Lauwers, 2003; Kirk, 2003).\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eRationale for the role of autophagy: Early dendritic and axonal dystrophy, reduction of striatal dopamine content, and the formation of somatic and dendritic ubiquitinated inclusions in DA neurons were prevented by ablation of Atg7 (an essential autophagy related gene (Friedman et al. 2012)).\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eRationale for the role of Ubiquitin Proteosomal System/Autophagic Lysosomal Pathway (UPS/ALP): Protection from DA neuronal death was also observed in multiple experiments through the pharmacological modulation of the UPS, ALP system; however, there are also contradicting data in the literature. (Inden et al. 2007; Fornai et al. 2003; Dehay et al. 2010; Zhu et al. 2007, Fornai et al. 2005).\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eHowever, although many lines of evidence exist to support essentiality of impaired proteostasis, a single molecular chain of events cannot be established.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE4\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eDegeneration of DA neurons of nigrostriatal pathway\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMODERATE\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eReceptors for advanced glycated end product (AGEs) can activate NF-kB (a transcription factor involved in the inflammatory response) and they are found on microglia cells and astrocytes. Ablation of receptor for advanced glycated end product (RAGE) proved to be protective against MPTP-induced decreases of TH\u003csup\u003e+ \u003c/sup\u003eneurons and mitigation of microglia and astrocytes reactivity was observed (Teismann et al. 2012).\u0026nbsp;Inhibition of RAGE, which is upregulated in the striatum following rotenone exposure and in response to neuroinflammation, decreases rotenone-induced apoptosis by suppressing NF(Nuclear Factor)-kB activation, as well as the downstream inflammatory markers TNF-alpha, iNOS and myeloperoxidase (Abdelsalam and Safar, 2015). This showed intermingled links between neuronal injury/death and neuroinflammation. Rotenone-induced neurotoxicity was less pronounced in neuron-enriched cultures than in neuron-glia co-cultures (Gao et al., 2002), suggesting that neuron-glia interactions are critical for rotenone-induced neurodegeneration. In addition, in in vitro systems, a decrease in thyroxine hydrosilase (TH) mRNA expression has been observed to be a sufficient signal to trigger microglial reactivity (Sandstr\u0026ouml;m et al., 2017).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE5 \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eNeuroinflammation\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMODERATE\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: Following treatment with Rotenone or MPTP/ MPP+, protection of DA neurons and terminals was observed in vivo and in vitro by inhibiting different feature of neuroinflammation (microglia/astrocyte); however, inhibition was different in different models and considered as an indirect evidence of essentiality (Zhou et al., 2007; Gao et al., 2002 and 2003 and 2015;\u0026nbsp;; Emmrich et al., 2013; Salama et al., 2012; Chang et al., 2013; Wang et al., 2014; Liu et al., 2012, 2015; Borrajo et al., 2013; Brzozowski et al., 2015; Wang et al., 2006; Chung et al., 2011; Sriram et al., 2014; Feng et al., 2002; Sathe et al., 2012; Khan et al., 2014; Ros-Bernal et al., 2011; Ferger et al., 2004; Chao et al., 2009; Rojo et al., 2010; Qian et al., 2011; Dehmer et al., 2000; Bodea et al., 2014). Mice lacking the type-1 Interferons receptor showed an attenuated pro-inflammatory response and reduced loss of dopaminergic neurons induced by MPTP/MPP\u003csup\u003e+\u003c/sup\u003e. The neuro-protective potential was also confirmed by treatment with a blocking monoclonal antibody against type-1A IFN receptor (interferon receptor) that increased survival of dopaminergic neurons of TH+ (Main et al., 2016).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE4\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eDegeneration of DA neurons of nigrostriatal pathway\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: Clinical and experimental evidences show that the pharmacological replacement of the dopamine (DA) neurofunction by allografting fetal ventral mesencephalic tissues is successfully replacing degenerated DA neurons resulting in the total reversibility of motor deficit in animal model and partial effect is observed in human patient for PD (Widner et al., 1992; Henderson et al., 1991; Lopez-Lozano et al., 1991; Freed et al., 1990; Peschanski et al., 1994; Spencer et al., 1992).\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eAlso, administration of L-DOPA or DA agonists results in an improvement of motor deficits (Calne et al 1970; Fornai et al. 2005). The success of these therapies in man as well as in experimental animal models clearly confirms the causal role of dopamine depletion for PD motor symptoms ( Connolly et al., 2014; Lang et al., 1998; Silva et al., 1997; Cotzias et al., 1969; Uitti et al., 1996; Ferrari-Tonielli et al., 2008; Kelly et al., 1987; Walter et al., 2004; Narabayashi et al., 1984; Matsumoto et al., 1976; De Bie et al., 1999; Uitti et al., 1997; Scott et al., 1998; Moldovan et al., 2015; Deuschl et al., 2006; Fasano et al., 2010; Castrito et al., 2011; Liu et al., 2014; Widner et al., 1992; Henderson et al., 1991; Lopez-Lozano et al., 1991; Freed et al., 1990; Peschanski et al., 1994; Spencer et al., 1992).\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eFurthermore, experimental evidence from animal models of PD and from in-vitro systems indicate that prevention of apoptosis through ablation of BCL-2 family genes prevents or attenuates neurodegeneration of DA neurons (Offen D et al., 1998; Dietz GPH et al. 2002).\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003ch3\u003e\u0026nbsp;\u003c/h3\u003e\r\n","weight_of_evidence_summary":"\u003ch4\u003eConcordance of dose-response relationship.\u003c/h4\u003e\r\n\r\n\u003cp\u003eData from experiments with the stressor compounds rotenone and MPTP (known inhibitors of the mitochondrial Complex I (CI)) reveal a good concordance of the dose-response relationships between the MIE and AO and within KEs. Although the different KEs have been measured using different methodologies, comparison of data from multiple in-vitro/in-vivo studies shows a general agreement in dose-relationship (see table 1 and 2). There is a good consistency when comparing data on KE4 and the AO after exposure to rotenone and MPTP. However, in vivo rodent studies proved that only exposure to low concentrations of rotenone (rat brain concentration between 20-30 nM of rotenone; Betrabet et al., 2000) or MPTP (mice striatum concentration of approximately 12-47 \u0026micro;M MPP+; Fornai et al., 2005; Thomas et al. 2012) after chronic exposure (approximately 5 weeks) reproduced the anatomical, neurochemical behavioural and neuropathological features similar to the ones observed in Parkinson\u0026rsquo;s disease (PD). Because of the variability of experimental protocols used, a clear no-effect threshold could not be established; nevertheless, these brain concentrations of rotenone (20-30 nM) and MPP+ (approximately 12-47\u0026micro;M) could serve as probabilistic thresholds for chronic exposure that could reproduce features of PD as both concentrations trigger approximately a 50% inhibition of Complex I (see table 3). Generally, a strong response-response relationship is observed within studies. Some exceptions for this rule are observed between KE3/KE5 and KE4, likely because of the all biological complexity associated with these KEs. In this AOP, neuroinflammation was considered to have a direct effect on degeneration of DA neurons. However, it was not clear at which conditions it would become a modulatory factor and for practical reasons was not included in table 1, 2 and 3 but considered in the weight of evidence analysis.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eTable1 Dose-response and temporality table for rotenone.\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eRotenone Concentration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eKE1\u003csup\u003eaaa\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eInhibition of C I\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eKE2\u003csup\u003eaaa\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eMitochondrial dysfunction\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eKE3\u003csup\u003eaaa\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eImpaired proteostasis\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eKE4\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eDegeneration of DA neurons of nigrostriatal pathway\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eAO\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eParkinsonian motor symptoms\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e5-10 nM \u003cem\u003ein-vitro\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e[1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4-72 hours [1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4-72 hours [4]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e24 hours [3]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e-\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e-\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e20-30 nM \u003cem\u003eex-vivo\u003c/em\u003e, rat brain concentration\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e[4-5-2-6]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4-72 hours (4-5)\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4-72 hours [4-5]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e24 hours [3-2-6]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e++\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e5 weeks [2-6]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003csup\u003eaa\u003c/sup\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e5 weeks [2-6]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e100 nM \u003cem\u003ein-vitro\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e[4]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4-72 hours [4]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4-72 hours [4]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e24 hours [3]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003eCorresponding to a concentration above the maximum tolerated dose in-vivo [2-6]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003eCorresponding to a concentration above the maximum tolerated dose in vivo [2-6]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003eReferences: Choi et al. 2008 [1]; Betarbet et al. 2006 [2]; Chou et al. 2010 [3]; Barrientos and Moraes 1999 [4]; Okun et al. 1999 [5]; Betarbet et al. 2000 [6]\u003c/p\u003e\r\n\r\n\u003cp\u003e-no data available\u003c/p\u003e\r\n\r\n\u003cp\u003e+: low severity score, ++ intermediate severity score, +++ high severity score\u003c/p\u003e\r\n\r\n\u003cp\u003ea: 50% of treated animals showed loss of DA neurons in SNpc\u003c/p\u003e\r\n\r\n\u003cp\u003eaa: All animals affected in KE4 showed impaired motor symptoms\u003c/p\u003e\r\n\r\n\u003cp\u003eaaa: KE 1, 2 and 3 showed a dose-related severity in the effect and the score ++ was normalized vs. the KE4\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eTable 2. Dose-Response and Temporality table for MPTP/MPP\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eMPTP Administered Dose\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eMPP\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eBrain Concentration\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE1\u003csup\u003ebb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eInhibition of C I\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE2\u003csup\u003ebb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eMitochondrial dysfunction\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE3\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eImpaired proteostasis\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE4\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eDegeneration of DA neurons of nigrostriatal pathway\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eAO\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eParkinsonian motor symptoms\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e1 mg/kg sc infusion [1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e-\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e-\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e-\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4 weeks[1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+\u003csup\u003eaaa\u003c/sup\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4 weeks [1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003eNo effect\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e5 mg/kg sc infusion [1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e-\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e-\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e-\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4 weeks[1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e++\u003csup\u003eaa\u003c/sup\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4 weeks [1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4 weeks [1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e20-30 mg/kg sporadic ip. injections (4 times every 2 hours)\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e[2, 1]\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e47\u0026micro;M [2]^\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e12\u0026micro;M [1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4 hrs [2]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4hrs [2]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4 weeks [1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e1-4 weeks[2,1]\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e+++\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e4 weeks [1]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nReferences. Fornai et al. 2005 [1]; Thomas et al. 2012 [2]\u003c/p\u003e\r\n\r\n\u003cp\u003e-no data available\u003c/p\u003e\r\n\r\n\u003cp\u003ea: approx 50% loss of DA neurons in SNpc\u003c/p\u003e\r\n\r\n\u003cp\u003eaa: approx 30% loss of DA neurons SN pc\u003c/p\u003e\r\n\r\n\u003cp\u003eaaa: no loss of DA neurons in SN pc. Reduced level of striata DA\u003c/p\u003e\r\n\r\n\u003cp\u003eb: for KE3, a dose response effect was observed.\u003c/p\u003e\r\n\r\n\u003cp\u003ebb: for KE 1 and 2 the severity of the effect was normalized vs. the KE4\u003c/p\u003e\r\n\r\n\u003cp\u003e^ After single dose MPTP administration, brain concentration was approx. 5.15 \u0026micro;M\u003c/p\u003e\r\n\r\n\u003ch4\u003eTemporal concordance among the MIE, KEs and AO.\u003c/h4\u003e\r\n\r\n\u003cp\u003eThere is a strong agreement that loss of DA neurons of the SNpc that project into the putamen is preceded by reduction in DA and degeneration of DA neuronal terminals in the striatum (Bernheimer et al. 1973). The clinical symptoms of a motor deficit are observed when 80% of striatal DA is depleted (Koller et al. 1992) and the sequence of pathological events leading to the adverse outcome has been well-documented (Fujita, et al.2014; O\u0026rsquo;Malley 2010, Dexter et al. 2013). Temporal concordance (see table 1 and 2) among the KEs can be observed in the experimental models of PD using the chemical stressors rotenone and MPTP (Betarbet 2000 and 2006; Sherer et al. 2003, Fornai et al. 2005). The acute administration of the chemical stressors can trigger a dose-related change from the MIE to impaired proteostasis; however, to trigger KE4 (i.e. degeneration of DA neurons in SNpc with presence of intracytoplasmatic Lewy-like bodies) and motor deficits (AO), proteostasis needs to be disturbed for a minimum period of time (Fornai et al. 2005).\u003c/p\u003e\r\n\r\n\u003ch4\u003eStrength, consistency, and specificity of association of AO and MIE.\u003c/h4\u003e\r\n\r\n\u003cp\u003eStrength and consistency of the association of the AO with the MIE is strong. There is a large body of evidence from in-vitro and in-vivo studies with chemical stressors, showing association between the MIE that triggers an inhibition of CI and the AO (Sherer et al. 2003; Betarbet et al. 2000 and 2006, Fornai et al. 2005). Human data also suggest a link between inhibition of CI and AO (Greenamyre et al. 2001; Schapira et al. 1989; Shults, 2004). Using the two different chemical stressors, rotenone and MPTP, data are consistent and the pattern of activation of the MIE leading of the AO is similar. For rotenone and MPTP, specificity is high; however, there are many inhibitors of the mitochondrial CI without evidence of triggering the AO. When considering \u0026nbsp;the limited amount of chemical stressors for which empirical data are available for supporting the full sequence of KEs, kinetic and metabolic considerations should be taken into account to demonstrate specificity for these compounds. The issue of specificity was also debated during the external review of this AOP and the following information was added:\u003c/p\u003e\r\n\r\n\u003cp\u003eThe vast majority of empirical support available in the literature is based on complex I inhibitors, such as rotenone and MPTP/MPP+, as well as on studies involving genetic impairment of complex I activity. A relatively wide spectrum of structurally different complex I inhibitors have been described over the course of recent decades. Prominent examples are acetogenins (Nat Prod Rep 2005, 22, 269-303); alkaloids (J Neurochem 1996, 66, 1174-1181); antibiotics (BBA 1998, 1364, 222-235; Eur J Biochem 1994, 219, 691-698; JBC 1970, 245, 1992-1997; Bioorg Med Chem 2003, 11, 4569-4575); pesticides (Biochem Soc Trans 1994, 22, 230-233); quinones (JBC 1971, 246, 2346-2353); or vanilloids (ABB 1989, 270, 573-577). Additional information can be also retreived from Fato et al 2009, Espositi et al. 1993, Lagoa et al. 2011 and Park et al. 2003.\u003c/p\u003e\r\n\r\n\u003cp\u003eAll of these structurally different complex I inhibitors were characterized with isolated mitochondria or with submitochondrial particles. Application of bovine heart mitochondria revealed IC\u003csub\u003e50\u003c/sub\u003e values in the range of 20-70 nM for piericidin A, fenpyroximate, rotenone, and phenoxan (Eur. J. Biochem 1994, 219, 691-698). IC\u003csub\u003e50\u003c/sub\u003e values in the range of 1-10 nM were detected by application of submitochondrial particles with rotenone, molvizarin, rollinstatin-1 and -2, and piericidin A (Biochem J. 1994, 301, 161-167).\u003c/p\u003e\r\n\r\n\u003cp\u003eStudies involving neuronal cell cultures or in vivo models are in fact rather rare. A systematic comparison of the IC\u003csub\u003e50\u003c/sub\u003e values for complex I inhibition and EC\u003csub\u003e50\u003c/sub\u003e values for the reduction of ATP levels; cell death was performed with rat fetal striatal neurons (Exp Neurol 2009, 220, 133-142). Due to the lipophilicity of most of the complex I inhibitors tested, the detected EC\u003csub\u003e50\u003c/sub\u003e values were in most cases lower than the IC\u003csub\u003e50\u003c/sub\u003e values detected for complex I inhibition. EC\u003csub\u003e50\u003c/sub\u003e values detected were: annonacin (60 nM), fenazaquin (45 nM), piericidin A (1.6 nM), rollinstatin- 2 (1 nM), rotenone (8 nM), and squamocin (1 nM).\u003c/p\u003e\r\n\r\n\u003cp\u003eA systematic investigation involving mesencephalic cultures as well as rats was performed for the complex I inhibitor annonacin, a major acetogenin of soursop, a plant suspected to cause an atypical form of PD in Guadeloupe. Mesencephalic cultures treated for 24 h with annonacin revealed EC\u003csub\u003e50\u003c/sub\u003e values of 20 nM (annonacin), 34 nM (rotenone), and 1900 nM (MPP\u003csup\u003e+\u003c/sup\u003e) (Neurosci 2003, 121(2), 287-296). Intravenous application by minipumps over the course of 28 days indicated a passage of annonacin across the blood-brain barrier, and an energy-dependent loss of ca. 30 % of DA neurons in the substantia nigra (Champi et al.2004)).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003ch4\u003eWeight of Evidence (WoE).\u003c/h4\u003e\r\n\r\n\u003ch4\u003eBiological plausibility, coherence, and consistency of the experimental evidence.\u003c/h4\u003e\r\n\r\n\u003cp\u003eThe biological plausibility of this AOP is overall considered strong. When using multiple stressors in different studies and assays, the coherence and consistency of the experimental data is well established. Furthermore, in-vivo and in-vitro studies are also in line with the human evidence from PD patients. In addition, although the mechanistic understanding of parkinsonian disorders (and PD in particular) are not fully clear, the KEs and KERs described in this AOP are considered critical for the development of the disease (Fujita et al. 2015, Shulman et al. 2011, Dexter et al. 2013, Dauer et al. 2003).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003e1 Support for Biological Plausibility of KERs\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eDefining Question\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eHigh (Strong)\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eModerate\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eLow(Weak)\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eIs there a mechanistic (i.e. structural or functional) relationship between KEup and KE down consistent with established biological knowledge?\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eExtensive understanding of the KER based on extensive previous documentation and broad acceptance\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eThe KER is plausible based on analogy to accepted biological relationships, but scientific understanding is not completely established\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eThere is empirical support for a statistical association between KEs but the structural or functional relationship between them is not understood\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eMIE =\u0026gt; KE1\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eBinding of inhibitor to NADH-ubiquinone oxidoreductase leads of complex I\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: As describe in this KER there is an extensive understanding of the functional relationship between binding of an inhibitor to NADH-ubiquinone oxidoreductase (CI) and its inhibition. Different complex I ligands, both naturally occurring, like rotenone (from Derris scandens), piericidin A (from Streptomyces mobaraensis), acetogenins (from various Annonaceae species) and their derivatives, and synthetically manufactured like pyridaben and various piperazin derivatives inhibit the catalytic activity of complex I (Degli Esposti, 1994: Ichimaru et al. 2008; Barrientos and Moraes, 1999; Betarbet et al., 2000).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE1 =\u0026gt; KE2\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eInhibition of complex I leads to mitochondrial dysfunction\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: There is extensive understanding of the mechanisms explaining how the inhibition of complex I lead to mitochondrial dysfunction (i.e. failure to produce ATP, increase in production of ROS etc). It is well documented that CI inhibition is one of the main sites at which electron leakage to oxygen occurs resulting in oxidative stress (Efremov and Sazanow, 2011; lauren et al. 2010; Greenamyre et al. 2001). These pathological mechanisms are well studied as they are used as readouts for evaluation of mitochondrial dysfunction (Graier et al., 2007; Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE2 =\u0026gt; KE3\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eMitochondrial dysfunction results in impaired proteostasis\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMODERATE\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: The weight of evidence supporting the biological plausibility behind the relationship between mitochondrial dysfunction and impaired proteostasis, including the impaired function of UPS and ALP that results in decreased protein degradation and increase protein aggregation is well documented but not fully understood. It is well established that the two main mechanisms that normally remove abnormal proteins (UPS and ALP) rely on physiological mitochondrial function. The role of oxidative stress, due to mitochondrial dysfunction, burdens the proteostasis with oxidized proteins and impairs the chaperone and the degradation systems. This leads to a vicious circle of oxidative stress inducing further mitochondrial impairment (Powers et al., 2009; Zaltieri et al., 2015; McNaught and Jenner, 2001). Therefore, the interaction of mitochondrial dysfunction and UPS /ALP deregulation plays a pivotal role in the pathogenesis of PD (Dagda et al., 2013; Pan et al., 2008; Fornai et al., 2005; Sherer et al., 2002).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE2 =\u0026gt; KE4\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eMitochondrial dysfunction leads to the degeneration of dopaminergic neurons of the nigrostriatal pathway\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: Mitochondrial are essential for ATP production, ROS management, calcium homeostasis and control of apoptosis. Mitochondrial homeostasis by mitophagy is also an essential process for cellular maintenance (Fujita et al. 2014). Because of their anatomical and physiological characteristics, SNpc DA neurons are considered more vulnerable than other neuronal populations (Sulzer et al. 2013; Surmeier et al.2010). Mechanistic evidence of mutated proteins relate the mitochondrial dysfunction in familial PD with reduced calcium capacity, increased ROS production, increase in mitochondrial membrane permeabilization and increase in cell vulnerability (Koopman et al. 2012; Gandhi et al. 2009). Human studies indicate mitochondrial dysfunction in human idiopathic PD cases in the substantia nigra (Keeney et al., 2006; Parker et al., 1989, 2008; Swerdlow et al., 1996). In addition, systemic application of mitochondrial neurotoxicants such as rotenone or MPTP leads to a preferential loss of nigrostriatal DA neurons (Langston et al., 1983).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE3 =\u0026gt; KE4\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eImpaired proteostasis leads to degeneration of DA neurons of the nigrostriatal pathway\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMODERATE\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: It is well known that impaired proteostasis refers to misfolded and aggregated proteins including alfa-synuclein, deregulated axonal transport of mitochondria and impaired trafficking of cellular organelles. Evidences are linked to PD and experimental PD models as well as from genetic studies (McNaught et al. 2001, 2003; Tieu et al. 2014; Arnold 2011; Rappold et al. 2014). Strong evidence for degeneration of the nigrostriatal pathway comes from the experimental manipulations that directly induce the same disturbances of proteostasis as observed in PD patients (e.g. viral mutated alpha-synuclein expression) or in chronic rotenone/MPTP models trigger degeneration of the nigrostriatal pathway (Kirk et al. 2003; Betarbet et al. 2000 and 2006; Fornai et al. 2005). However, a clear mechanistic proof for the understanding of the exact event triggering cell death is lacking. There is only moderate evidence showing that interventions that correct disturbances of proteostasis after exposure to rotenone would prevent neuronal degeneration and that the disturbances of proteostasis correlate quantitatively under many conditions with the extent of nigrostriatal neuronal degeneration.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE4\u0026nbsp;=\u0026gt; KE5\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eDegeneration of DA neurons of the nigrostriatal pathway leads to neuroinflammation\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMODERATE\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: The fact that neuronal injury/death can trigger neuroinflammation is supported by evidence in human and experimental models. The evidence that neuroinflammation triggered by neuronal damage can cause neuronal death (vicious circle), is mostly indirect (blockade of any feature of neuroinflammation) or by analogy (Hirsch and Hunot, 2009; Tansey and Goldberg, 2009; Griffin et al., 1998; McGeer and Mc Geer, 1998; Blasko et al., 2004; Cacquevel et al., 2004; Rubio-Perez and Morillas-Ruiz, 2012; Thundyil and Lim, 2014; Barbeito et al., 2010).\u0026nbsp;Neuroinflammation is observed in idiopathic and in genetic human PD as well as in complex I inhibitor exposed humans, non-human primates, and rodent. Components of damaged neurons lead to glial cells activation via Toll-like receptors. Several chemokines and chemokine receptors (fraktalkine, CD200) control the neuron-microglia interactions. Neuroinflammation in response to damaged neurons is not confined to PD, but is common to several neurodegenerative diseases\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE5 =\u0026gt; KE4\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eNeuroinflammation leads to degeneration of DA neurons of the nigrostriatal pathway\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMODERATE\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: The fact that reactive glial cells (microglia and astrocytes) may kill neurons is well accepted. The mechanisms underlying this effect may include the release of cytotoxic signals (e.g. cytokines) or production of ROS and RNS (Chao et al., 1995\u0026nbsp;; Brown and Bal-Price, 2003\u0026nbsp;; Kraft and Harry, 2011\u0026nbsp;; Taetzsch and Block, 2013). However, the responsible mediators differ from model to model.\u0026nbsp;In humans or non-human primates, an inflammatory activation of glial cells is observed years after exposure to complex I inhibitors. Activated microglia and astrocytes form pro-inflammatory cytokines and free radical species, mostly responsible for neuronal damage. Glial reactivity promotes an impairment of blood brain barrier integrity, allowing an infiltration of peripheral leukocytes that exacerbate the neuroinflammatory process and contribute to neurodegeneration.The debris of degenerating neurons causes neuroinflammation, which in turn can trigger neurodegeneration, thus leading to a self-perpetuating vicious cycle.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE4\u0026nbsp;=\u0026gt; AO\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eDegeneration of DA neurons of the nigrostriatal pathway leads to parkinsonian motor symptoms\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: The mechanistic understanding of the regulatory role of striatal DA in the extrapyramidal motor control system is well established. The loss of DA in the striatum is characteristic of all aetiologies of PD and is not observed in other neurodegenerative diseases (Bernheimer et al. 1973; Reynolds et al. 1986). Characteristic motor symptoms such as bradykinesia, tremor, or rigidity are manifested when more than 80\u0026nbsp;% of striatal DA is depleted as a consequence of SNpc DA neuronal degeneration (Koller et al. 1992).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003ch4\u003eEmpirical support.\u003c/h4\u003e\r\n\r\n\u003cp\u003eEmpirical support is strong. Many studies show evidence for the KERs by showing temporal concordance and dose concordance when using different stressors.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003e3 Empirical support for KERs\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eDefining Question\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eDoes the empirical evidence support that a change in the KEup leads to an appropriate change in the KE down? Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup higher than that for KE down?\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eAre inconsistencies in empirical support cross taxa, species and stressors that don\u0026rsquo;t align with expected pattern of hypothesized AOP?\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eHigh (Strong)\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eModerate\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eLow(Weak)\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMultiple studies showing dependent change in both exposure to a wide range of specific stressors (extensive evidence for temporal, dose-response and incidence concordance) and no or few critical data gaps or conflicting data.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eDemonstrated dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories, etc.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eLimited or no studies reporting dependent change in both events following exposure to a specific stressor (ie endpoints never measured in the same study or not at all); and/or significant inconsistencies in empirical support across taxa and species that don\u0026rsquo;t align with expected pattern for hypothesized AOP\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eMIE =\u0026gt; KE1 \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eBinding of inhibitor to NADH-ubiquinone oxidoreductase leads to partial or total inhibition of complex I\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: The inhibition of complex I is well documented in a variety of studies using isolated mitochondria or cells as well as in in vivo experiments and in human post mortem PD brains. In many experiments using different inhibitors ie rotenone and MPTP, the observed relationship between the two events was temporal, response and dose concordant (Betarbet et al., 2000 and 2006, Okun et al., 1999, Koopman et al., 2007, Choi et al., 2008, Grivennikova et al., 1997, Barrientos and Moraes 1999).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE1 =\u0026gt; KE2 \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eInhibition of complex I leads to mitochondrial dysfunction\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: There is a large amount of studies showing that the inhibition of CI inhibition results in mitochondrial dysfunctions in a response and dose dependent manner (Barriento and Moraes, 1999).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE2 =\u0026gt; KE3 \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eMitochondrial dysfunction results in impaired proteostasis\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: Based on the existing in vitro and in vivo data it is suggested that mitochondrial dysfunction impairs protein homeostasis (impairment of the UPS and ALP system) through oxidative and nitrosative stress resulting in accumulation of misfolded proteins (including \u0026alpha;-synuclein), disruption of microtubule assembly and damaged intracellular transport of proteins and cell organelles. A number of studies performed with chemical stressors showed evidence of temporal, response and dose concordance (Chou et al. 2010; Betarbet et al. 2000 and 2006; Fornai et al. 2005).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE2 =\u0026gt; KE4\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eMitochondrial dysfunction directly leads to degeneration of DA neurons of nigrostriatal pathway\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: Multiple \u003cem\u003ein vitro\u003c/em\u003e studies indicate dose and response-response concordance. As most of the studies were conducted \u003cem\u003ein vitro\u003c/em\u003e, the temporal concordance is difficult to establish; however, can be expected based on the well know temporal sequence of the two KEs. (Park et al., 2014; Choi et al., 2014; Marella et al., 2008; Du et al. 2010; Hajieva et al., 2009; Sherer et al., 2003; Sherer et al., 2007; Wen et al. 2011; Swedlow et al., 1996; Jana et al., 2011; Jha et al., 2000; Chinta et al., 2006)\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE3 =\u0026gt; KE4a\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eImpaired proteostasis leads to degeneration of DA neurons of the nigrostriatal pathway\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: The empirical support linking impaired proteostasis with degeneration of DA neurons of the nigrostriatal pathway is strong and comes from in-vivo and in-vitro studies performed with different stressor (i.e. Rotenone, MPTP or proteasome inhibitors) and post-mortem human evidences in PD patients supporting a causative link between the two key events. Temporal, effect and dose concordance was established in a number of experiments (Fornai et al. 2005; Fornai et al. 2003; Betabret et al. 2000 and 2006).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE4a =\u0026gt; KE5\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eDegeneration of DA neurons of nigrostriatal pathway leads to neuroinflammation\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMODERATE\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: multiple in vivo and in vitro experiments support the link between degeneration of DA neurons in the nigrostriatal pathway and neuroinflammation. The observation of concomitant presence of reactive microglial and astrocytic cells and degenerated/degenerating DA neurons is also reported in many studies with a good temporal and response concordance. ATP and other damage associated molecular patterns (DAMPs), released from degenerating cells, stimulate P2Y receptors on microglia, leading to their activation. Experimental injection of DAMPs, fraktalkine, or neuromelanin, released by degenerating DA neurons evokes neuroinflammation. Neutralization of DAMPs (e.g. antibodies against HMGB1 or CX3CR1) decreases MPTP-induced neuroinflammation. Toll-like receptor 4 deficient mice display a reduced neuroinflammatory response upon MPTP treatment. Inhibition of RAGE, which is upregulated in striatum upon rotenone exposure, suppresses NF-kB activation and downstream inflammatory markers.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE5 =\u0026gt; KE4b\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eNeuroinflammation leads to degeneration of DA neurons of nigrostriatal pathway.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMODERATE\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: multiple in vivo and in vitro experiments support the link between neuroinflammation and degeneration of DA neurons in the nigrostriatal pathway. The observation of concomitant presence of reactive microglial and astrocytic cells and degenerated/degenerating DA neurons is also reported in many studies with a good temporal and response concordance. Neuroinflammation has been implicated in dopaminergic neuronal cell death in PD patients (Vivekanantham et al., 2014).\u0026nbsp;LPS injection into the CNS, or applied systemically, evokes glial inflammation and a preferential degeneration of DA neurons. In mouse models with a knockout of either IL-1b, IFN-g, or TNF-a receptors 1 and 2, LPS no longer evokes neuroinflammation and DA neurodegeneration. Experimental interference with CD4+ T cell activation protects from DA neurodegeneration. Transfer of immunosuppressive regulatory T cells protect from DA neurodegeneration. Anti-inflammatory TGF-b1 signaling protects from DA neurodegeneration. Clinical trials indicate a protective influence on DA neuron survival by the antibiotic minocycline blocking microglial reactivity, in association with rasagiline (prevents DA degeneration), and coenzyme Q10/creatine (restoration of cellular ATP).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKE4b\u0026nbsp;=\u0026gt; AO \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eDegeneration of DA neurons of nigrostriatal pathway leads to parkinsonian motor symptoms\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSTRONG\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003eRationale: The experimental support linking the degeneration of DA neurons of nigrostriatal pathways with the manifestation of motor symptoms of PD comes from human in vivo observations as well as from monkey, mice and rat in vivo models exposed to an experimental toxin i.e. rotenone and MPTP. Observations in human allow defining correlation between the levels of striatal DA with the onset of motor dysfunction (Lloyd et al. 1975; Hornykiewicz et al. 1986; Bernheimer et al. 1973). Temporal, effect and dose concordance comes from studies performed in multiple animal species following administration of rotenone and MPTP (Bezard et al. 2001; Cannon et al. 2009; Petroske et al. 2001; Alvarez-Fischer et al. 2008; Blesa et al. 2012; Lloyd et a. 1975).\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003ch4\u003eUncertainties and Inconsistencies.\u003c/h4\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThe strength of this AOP is mainly based on MPP+ and rotenone and only limited information on whether other mitochondrial complex I inhibitors also perturb the KEs (specifically degeneration of DA neurons in the SNpc) \u0026nbsp;or produce a similar AO.\u003c/li\u003e\r\n\t\u003cli\u003eConflicting data exists (Choi et al. 2008) showing that mitochondrial complex I inhibition is not required for DA neuron death induced by rotenone, MPTP/MPP+, or paraquat,\u0026nbsp;challenging\u0026nbsp;the current AOP. The cited research article shows that abolishment of complex I\u0026rsquo;s activity by inactivation of a gene that codes for a subunit of complex I does not impact the survival of DA neurons in culture. The actions of rotenone, MPTP/MMP+ are independent of complex I. Since some complex I inhibitors also target other complexes, it is possible that impairment of other respiratory complexes may be involved.\u0026nbsp;It was noted that this paper used the approach of genetically deleting an essential chaperone in complex I assembly, and the authors found that absence of complex I activity in this model did not affect the toxicity of rotenone and MPP+. However, the findings have never been confirmed/ continued, neither in the originating laboratory, nor by others. Second, the work did not consider the possibility that some functions of complex I were not affected by the absence of the chaperone (e.g. reverse electron transfer from complex II and III), and that rotenone and MPTP/MPP+ may well cause toxicity by interfering with such residual function (e.g. by favoring channeling of electrons to molecular oxygen). In light of this situation, the publication of Choi et al (2008) should be considered weak in the overall weight of evidence and therefore considered\u0026nbsp;a minor inconsistency.\u0026nbsp;\u003c/li\u003e\r\n\t\u003cli\u003eThere is no strict linear relationship between inhibitor binding and reduced mitochondrial function. Low doses of rotenone that inhibit CI activity partially do not alter mitochondrial oxygen consumption. Therefore, bioenergetics defect cannot account alone for rotenone-induced neurodegeneration. Instead, under such conditions, rotenone neurotoxicity may result from oxidative stress (Betarbet et al., 2000). Few studies used human brain cells/human brain mitochondria. Therefore, full quantitative data for humans are not available.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eIt is molecularly unclear how rotenone binding alter CI function, switching it to ROS production. It is still unclear whether the site of superoxide production in CI inhibited mitochondria is complex I itself or not (Singer and Ramsay, 1994).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eSome studies suggest that rotenone and MPTP/MPP\u003csup\u003e+\u003c/sup\u003e may have effects other than CI inhibition, e.g. MPTP and rotenone can induce microtubule disruption (Feng, 2006; Ren et al., 2005; Cappelletti et al., 1999; Cappelletti et al., 2001, Brinkley et al., 1974; Aguilar et al., 2015).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThere are additional feedback possible between KEs, e.g. ROS production from KE2 may damage CI, this leads to enhancement of KE1.\u003c/li\u003e\r\n\t\u003cli\u003eSome KEs e.g. KE 2, 3, 5 pool molecular processes that may need to be evaluated individually at a later stage.\u003c/li\u003e\r\n\t\u003cli\u003eThe exact molecular link from mitochondrial dysfunction to disturbed proteostasis is still unclear (Malkus et al., 2009; Zaltieri et al., 2015).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThe role of ATP depletion vs. other features of mitochondrial dysfunction is not clear.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThe role of a \u0026alpha;-synuclein in neuronal degeneration is still unclear as well as the mechanisms leading to its aggregation.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eIt is not clear under which conditions KE3 and KE5 become modulatory factors, and when they are essential. MPTP/MPP\u003csup\u003e+\u003c/sup\u003e can induce damage to nigrostriatal neurons without formation of Lewy bodies (Dauer 2003; Forno 1986, 1993). Similarly, discontinuous administration of rotenone, even at high doses, damages the basal ganglia but produce no inclusions (Heikkila et al., 1985; Ferrante et al., 1997, Lapontine 2004). To reproduce the formation of neuronal inclusions, continuous sc infusion of MPTP/MPP\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;or rotenone is necessary. Acute intoxication with rotenone seems to spare dopaminergic neurons (Dauer et al., 2003, Ferrante 1997). In addition, in rats chronically infused with rotenone showed a reduction in striatal DARPP-32-positive (dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32,000), cholinergic and NADPH diaphorase-positive neurons (Hoglinger 2003) or in other brain regions. These results would suggest that Rotenone can induce a more widespread neurotoxicity (Aguilar 2015) or the model is not reproducible in all laboratories.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eInconsistent effects of MPTP/MPP+ on autophagy (up or down regulation) are reported (Drolet et al., 2004: Dauer et al., 2002). There is conflicting literature on whether increased autophagy would be protective or enhances damage. Similarly, a conflicting literature exists on extent of inhibition or activation of different protein degradation system in PD and a clear threshold of onset is unknown (Malkus et al., 2009; Fornai et al., 2005).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThe selective vulnerability of the SNpc dopaminergic pathway does not have a molecular explanation.\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003eIn some instances, the differential vulnerability of various brain regions towards a generalized complex I inhibition\u0026nbsp;found non-dopaminergic lesions, particularly in the striatum, in all animals with nigral lesion, as seen in atypical parkinsonism but not in idiopathic Parkinson\u0026#39;s disease (Hoglinger et al., 2003)\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003ePriority of the pattern leading to cell death could depend on concentration, time of exposure and species sensitivity; these factors have to be taken into consideration for the interpretation of the study\u0026rsquo;s result and extrapolation of potential low-dose chronic effect as this AOP refers to long-time exposure.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThe model of striatal DA loss and its influence on motor output ganglia does not allow to explain specific motor abnormalities observed in PD (e.g. resting tremor vs bradykinesia) (Obeso et al., 2000). Other neurotransmitters (Ach) may play additional roles. Transfer to animal models of PD symptoms is also representing an uncertainties.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThere are some reports indicating that in subacute rotenone or MPTP models (non-human primates), a significant, sometimes complete, recovery of motor deficits can be observed after termination of toxicant treatment. The role of neuronal plasticity in intoxication recovery and resilience is unclear.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThis AOP is a linear sequence of KEs. However, mitochondrial dysfunction (and oxidative stress) and impaired proteostasis are influencing each other and this is considered an uncertainties (Malkus et al., 2009).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003ch3\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e- Revision of AOP3 (Project:\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e \u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003ca href=\"https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms\" style=\"color:#467886; text-decoration:underline\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eNP/EFSA/PREV/2024/02\u003c/span\u003e\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e:\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/h3\u003e\r\n\r\n\u003cp\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eConcentration-response data for the prototypical stressors \u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003edeguelin, fenpyroximate, pyrimidifen, rotenone and tebufenpyrad\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e has been derived from Alimohammadi et al. 2023 Delp et al. 2019, 2021, van der Stel-OECD 2020, van der Stel et al. 2020 (Fig. 1).\u0026nbsp; Data points were extracted manually from graphs using PlotDigitizer (v3.0.0). The curve fits were generated using the \u003cem\u003eL.4\u003c/em\u003e function from the \u003cem\u003edrc\u003c/em\u003e package in R and organised across KEs to facilitate the analysis of the concordance of the concentration-response relationship. The different KEs have been measured in vitro.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cimg alt=\"\" src=\"https://aopwiki.org/system/dragonfly/production/2025/10/02/1gr52288he_E1N5VCglQdbK9ymD.png\" /\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003eFigure 1: Concentration-response data for the prototypical stressors deguelin, fenpyroximate, pyrimidifen, rotenone and tebufenpyrad across eight endpoints. All experiments were performed using cells cultured in glucose-containing medium.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cimg alt=\"\" src=\"https://aopwiki.org/system/dragonfly/production/2025/10/02/6klzkzi4o6_yEU6sCck4sUmi6f2.png\" /\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003eFigure 2: Concentration-response data for the prototypical stressors deguelin, fenpyroximate, pyrimidifen, rotenone and tebufenpyrad across five endpoints with cells cultured in galactose-containng medium.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eDeguelin\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e \u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eaffected KE 88\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e7 in both LUHMES and HepG2 cells at 10-3\u003c/span\u003e\u003c/span\u003e0 \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003enM in permeabilized cells. Oxygen consumption rate was affected at a similar concentration in intact cells (KE 177\u003c/span\u003e\u003c/span\u003e). ATP content, neurite \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003einte\u003c/span\u003e\u003c/span\u003egrity and viability were affected at ~ 10 \u0026micro;M when cultured in glucose containing\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e medium (Fig 1). When using galactose-containing medium, KE 177 and KE 890 were affected\u003c/span\u003e\u003c/span\u003e at concentrations similar to KE 887\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e (Fig2). \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRotenone \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eaffected KE 887 in both LUHMES and HepG2 cells at 60-100 nM\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e in permeabilized cells. Oxygen consumption rate and mitochondrial membrane potential \u003c/span\u003e\u003c/span\u003ewas\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e affected at a similar concentration in intact cells (KE 177) (Fig 1). ATP levels and cell viability was inhibited only at higher concentrations (4 \u0026ndash; 40 \u0026micro;M) when using glucose-containing medium\u003c/span\u003e\u003c/span\u003e. Interestingly, neurite integrity was already damaged at 20 \u0026ndash; 200 \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003enM\u003c/span\u003e\u003c/span\u003e, in line with KE 887 and KE 177, suggesting the contribution of additional mechanisms other than cI inhibitors. \u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eThis is in accordance with studies showing that rotenone inhibits microtubule assembly independently of a specific energy-requiring step (Brinkley et al. 1974; Marshall et al. 1978). This effect is particularly relevant because LUHMES cells are exposed at day 2 of differentiation, an early developmental stage characterised by neurite outgrowth, where microtubules dynamics play a critical role (Rieder et al. 1997, Dehmelt and Shelley)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eFor \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003ec\u003c/span\u003e\u003c/span\u003eells cultured in galactose-containing\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e medium\u003c/span\u003e\u003c/span\u003e, nanomolar rotenone concentrations were sufficient\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e to reduce ATP\u003c/span\u003e\u003c/span\u003e and viability (10-100 nM\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e)\u003c/span\u003e\u003c/span\u003e, with neurite integrity still being\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e more sensitive. \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eFenpyroximate \u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003einhibited KE 887\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e in HepG2 \u003c/span\u003e\u003c/span\u003eat ~ 20 \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003enM; no concentration-response data was available for LUHMES cells. KE 177 and KE 890 were affected at 10 \u0026ndash; 50 \u0026micro;M when using glucose-containing medium (Fig 1). \u003c/span\u003e\u003c/span\u003eWhen using galactose-containing medium, KE 177 and KE 890 were affected at concentrations similar to\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e KE 888 (Fig 2). \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003ePyrimidifen \u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003einhibited KE 888 in HepG2 at\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e ~7\u003c/span\u003e\u003c/span\u003e \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003enM\u003c/span\u003e\u003c/span\u003e; no concentration-response data was available for LUHMES cells\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e.\u003c/span\u003e\u003c/span\u003e KE 177 and KE 890 were affected at 16-50 \u0026micro;M when using glucose-containing medium (Fig\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e 1).\u003c/span\u003e\u003c/span\u003e In galactose-containing medium, only KE 890 was measured\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e, with \u003c/span\u003e\u003c/span\u003eEC50s for neurite integrity and viability of 3 and 30 nM, \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003ere\u003c/span\u003e\u003c/span\u003espectively (Fig\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e 2).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eTebufenpyrad \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003einhibited\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e KE 88\u003c/span\u003e\u003c/span\u003e8 in LUHMES \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003ecell\u003c/span\u003e\u003c/span\u003es at 40 nM\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e (\u003c/span\u003e\u003c/span\u003en = 1 biological\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e replicate)\u003c/span\u003e\u003c/span\u003e and in HepG2 at\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e 5.5 \u0026micro;M. \u003c/span\u003e\u003c/span\u003eIn intact LUHMES \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003ecells\u003c/span\u003e\u003c/span\u003e cultured in glucose-containing\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e medium\u003c/span\u003e\u003c/span\u003e, oxygen consumption rate was affected at 45 \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003en\u003c/span\u003e\u003c/span\u003eM, but mitochondrial membrane potential, ATP content and KE 890 only at\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e 10 \u0026ndash; 50 \u0026micro;M\u003c/span\u003e\u003c/span\u003e (Fig\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e 1)\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e. \u003c/span\u003e\u003c/span\u003eIn \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003egalactose-containing medium, \u003c/span\u003e\u003c/span\u003emitochondrial membrane \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003epotential\u003c/span\u003e\u003c/span\u003e was affected at 6 nM and ATP content between 6-10 nM\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e. \u003c/span\u003e\u003c/span\u003eEC50s for neurite integrity and viability \u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003ewere\u003c/span\u003e\u003c/span\u003e 10-40 and 130 \u0026ndash; 660 nM, respectively\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"color:black\"\u003e (Fig. 2).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Aptos\u0026quot;,sans-serif\"\u003eOverall cI inhinibitors show concordance in the concentrations response relationships across KEs\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eTable 2 Summary of quantitative effects of cI inhibitors\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cimg alt=\"\" src=\"https://aopwiki.org/system/dragonfly/production/2025/10/02/942rkfnpjv_Table_effects.jpg\" /\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003eUncertainties and inconsistencies table\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\" dir=\"ltr\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eUncertainty\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eImpact\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eReason\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eKE 888 measured in permeabilised cells\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePermeabilisation provides direct access for the tested compounds and substrates to the mitochondria and respiratory chain components. The physicochemical properties of the tested compound may reduce its ability to permeate the plasma membrane of intact cells, thus reducing or preventing its uptake which could affect the concentration and the time required to impact the downstream KEs.\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eLack of data in galactose condition\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eHigh\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eIn vitro cell models in general are characterized by an unphysiological reliance on glycolysis. In the presence of glucose any KE is influenced by the contribution of oxidative phosphorylation in addition to glycolisis to meet the cellular need for ATP. Thus, the KEs are influenced by the glycolisis rate. Glucose concentrations in culture medium higher than the physiological level enhances cellular resistance to mitochondrial dysfunction. Application of galactose instead of glucose in the medium allows a shift towards mitochondrial ATP generation. Even under these conditions, glycolysis significantly contributes to ATP production.\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eUse of HepG2 concentration response curves related to the measurement of oxygen consumption upon inhibition of cIII as a surrogate to represent inhibition of cIII in LUHMES cells, due to the lack of concentration response data for LUHMES cells\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eLow\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eIt is assumed that since the exposure is acute and in permeabilized cells, the test chemical would have immediate access to the mitochondria. Other mechanisms such as transport into the cells or an indirect effect via other signaling pathways were considered negligible under these assay conditions.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eBrain vs liver mitochondria\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e-\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eA study by Balmaceda et al. (2024) in isolated mitochondria provides evidence for intrinsic bioenergetic differences between brain and liver mitochondria obtained from mice, highlighting tissue-specific substrate preferences, redox states, and sensitivity to electron transport system (ETS) deficiencies. Their findings demonstrate that brain mitochondria rely more heavily on Complex I (CI) substrates and exhibit greater vulnerability to CI and Complex III (CIII) dysfunction, whereas liver mitochondria preferentially utilize Complex II (CII) substrates and show metabolic resilience (Balmaceda et al., 2024). These observations are supported by Lesner et al. (2022), who reported that CI is dispensable in liver but essential in brain tissue, and by Szibor et al. (2020), who confirmed higher ROS production in brain mitochondria under reverse electron transport (RET) conditions. Additionally, Rossignol et al. (1999, 2000) emphasized tissue-specific thresholds in oxidative phosphorylation (OXPHOS) control. By contrast, Gusdon et al. (2015) observed similar ETC enzyme activities in mitochondria from different tissues. However, they also confirmed a greater tendency for ROS production in brain mitochondria. This suggests that functional outcomes may be more dependent on systemic or regulatory factors than on the intrinsic properties of mitochondria. The differing results regarding the electron transport system may be due to the higher methodological resolution employed by Balmaceda et al. (2024), which included high-resolution respirometry and real-time coenzyme Q redox monitoring rather than bulk measurements of enzymatic activity and substrate transport. This approach permitted a more detailed and mechanistic understanding of ETS sensitivity and tissue-specific mitochondrial function.\u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eAdditional factors that can contribute to tissue-specific differences are mtDNA heteroplasmy and lineage-specific transcriptional networks established during development (Burr et al. 2023).\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eno concentration-response data for OCR in LUHMES\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eHigh\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eIncrease the uncertainty in the concordance concentration response relationship across the KEs\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eExposure is limited in concentrations and to 24 h\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eHigh\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eIt is possible that the effects on KE 887\u0026nbsp;and KE177 occur with higher potency or occur more rapidly than those required to observe an effect on KE890.\u0026nbsp;\u0026nbsp;The loss of temporal resolution may determine\u0026nbsp;an excessive steepness of the dose\u0026ndash;response curve.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eNeurite outgrowth assays (NA)\u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMedium\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTypically conducted from DoD2 to DoD3. NA tested on differentiating neurons is not representative of an adult stage. Active molecular mechanisms that are no longer present in adult or differentiated cells are involved in development. A reduction in NA area may be due to the degeneration of neurites or interference with developing pathways. In this exposure scenario, it is unclear whether the chemical would lead to a loss of neurons, or only a delay in neurite outgrowth. A loss in neurite integrity in the absence of a loss of viability was not considered sufficient to indicate activation of KE 890.\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eData reporting\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMedium - Low\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eFor some assays and chemicals, only two biological replicates were performed (instead of 3), therefore results should be considered with caution (see KERs empyrical evidence).\u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eFor some assay-chemical combinations it was unclear whether multiple studies repeated the same experiment, or if existing data was reprinted. If experiments were repeated and similar results obtained, this would indicate a higher confidence in the results. If results were simply re-printed, this can lead to an overestimated confidence. As the underlying raw data was not available, it was not possible to investigate further.\u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eDifferent assays have a different effect concentration (i.e. EC25 and EC50). Occasionally, also the same assay can have different effect levels depending on the publication, which reduces overall comparability. However, in most cases the EC25 and EC50 are within a factor of 3 of each other, thus limiting the uncertainty.\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eTemporal concordance across the AOP\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eKE887 is effective withinseconds and changes in MMP (KE177) can be detected within minutes. Time concordance could not be evaluated across KEs 177 and 890 since measurements were available at a single time point (24 h).\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e- Not endorsed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u0026nbsp;\u003c/p\u003e\r\n","quantitative_considerations":"\u003cp\u003eThe quantitative understanding of this AOP includes a clear response-response relationship and the identification of a threshold effect. The WoE analysis clearly supports the qualitative AOP as a means to identify and characterize the potential of a chemical to induce DA neuronal loss and the AO. Importantly, both the AO and the KE4 are considered relevant regulatory endpoints for this AOP. The empirical evidence supports existence of a response-response relationship. This response-response is likely triggered by a the brain concentrations of approximately 20-30 nM and 17-47 \u0026micro;M of rotenone and MPTP/MPP+ respectively and both concentrations trigger approx. a 50% inhibition of mitochondrial complex I and this could be considered as a \u0026ldquo;threshold\u0026rdquo;. However, a more detailed dose-response analysis for each KE is lacking as well as it is not clear which temporal relationship exists for lower CI inhibitory effects. It is clear from the analysis of the AOP that for the identification of these AOs, the design of the in-vivo studies should be tailored as to a MIE which leads to a long-lasting perturbation of the KEs. This provides the most specific and definite context to trigger neuronal death. To observe KEs relevant for this AOP, new endpoints need to be introduced. Although a dose, response and temporal relationship is evident for most KEs, the quantitative relationship between impaired proteostasis and degeneration of DA neurons has yet to be elucidated. Moving from a qualitative AOP to quantitative AOP would need a clear understanding of effect thresholds and this is still representing a major hurdle for several KEs of this AOP.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n\u003cstrong\u003eTable 3 Summary of quantitative effects at the concentration of rotenone and MPTP triggering the AO.\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eConcentration\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eKE1\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eInhibition of C I\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eKE2\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eMitochondrial dysfunction\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eKE3\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eImpaired proteostasis\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eKE4\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eDegeneration of DA neurons of nigrostriatal pathway\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eAO\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eParkinsonian motor symptoms\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eRotenone\u003c/strong\u003e 20-30 nM rat brain concentration\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e[1-2]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eApprox. \u003cstrong\u003e53%\u003c/strong\u003e[4-5]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eApprox. 20-53% (decrease in respiration rate)[1-2]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eApprox. 20-60% (decrease in UPS (26S) activity) [3]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eNeuronal loss (50% of animal affected) [2]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMotor impairment (100% of animals with neuronal loss) [2]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eMPP\u003c/strong\u003e+ 12-47 \u0026micro;M rat brain concentration [4-5]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eApprox. \u003cstrong\u003e50-75%\u003c/strong\u003e [5]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eApprox. 38% (reduction in phosphorylating respiration) [5]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eApprox. 60% (decrease in UPS activity) [4]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eApprox. 50% of neuronal loss [4-5]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMotor impairment [4]\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n[1]; Okun et al., 1999 [2]; Barrientos and Moraes 1999; [3] Borland et al., 2008 [4] Thomas et al., 2012; [5] Betarbet et al., 2000 and 2006.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n\u003cstrong\u003eSummary of the proposed Key Events in this AOP:\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003ca class=\"image\" href=\"https://aopwiki.org/wiki/index.php/File:Final_graph.jpg\"\u003e\u003cimg alt=\"Final graph.jpg\" src=\"https://aopwiki.org/wiki/images/c/c4/Final_graph.jpg\" style=\"height:495px; width:813px\" /\u003e\u003c/a\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eChronic, low level of exposure to environmental chemicals that inhibit complex I could result in mitochondrial dysfunction and oxidative stress, triggering proteasomal dysfunction strongly implicated in parkinsonian disorders, including aggregation/modifications in \u0026alpha;-synuclein protein and organelles trafficking. These cellular key events cause DA terminals degeneration in striatum and progressive cell death of DA neurons in SNpc, accompanied by neuroinflammation that potentiates neuronal cell death, finally leading to parkinsonian\u0026#39;s motor symptoms. Important to notice that at each step, the effects become regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e- \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003eRevision of AOP3 (Project:\u003c/strong\u003e \u003ca href=\"https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms\" style=\"-webkit-user-drag:none; -webkit-tap-highlight-color:transparent; user-select:text; font-variant-ligatures:normal; white-space:pre-wrap; color:inherit; cursor:text\" target=\"_blank\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#467886\"\u003eNP/EFSA/PREV/2024/02\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/a\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e: \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eA preliminary quantitative AOP has been published in Tebby et al. (2022). This qAOP was developed by modelling the KER using a set of mathematical functions, for two chemicals, rotenone and deguelin, based on data obtained in LUHMES cells.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eComplex I activity was measured using in proliferating LUHMES cells. Decrease in mitochondrial respiration was and measured using an Agilent\u0026reg; Seahorse OCR equipment. Mitochondrial respiration and proteasomal activity were measured using the same cells at a stage of neurite growth (day 3 of differentiation). The proteasomal function of cells was assessed at 24 h after toxicant exposure by a fluorogenic substrate that increases in fluorescence when the proteasome is active (Delp et al., 2021). Neuronal degeneration was represented by neurite area which was measured at a stage of neurite growth (day 2 of differentiation). The neurite areas (which serves as indirect measurement of neuronal interconnectivity) of stained differentiating neurons, as well as cellular viability were measured simultaneously at 24 h after toxicant exposure using high content imaging.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eEach KER was modelled a mathematical equation, either A) a 4-parameter log-logistic (Hill) function often used for dose-response modelling, B) a 2-parameter linear function which implies equal EC50 for the two adjacent KEs, or C) an increasing function which increases towards a horizontal asymptote, with an optional horizontal shift, implying a higher EC50 in the downstream KE. A ramification for KE3 was modelled: neurite area (KE4) was modelled as the product of two functions of mitochondrial respiration (KE2) and proteasomal activity (KE3) under the assumption that both KEs acted independently on it. The model parameters were estimated in a Bayesian statistical framework independently for rotenone and deguelin. The results are described in the open access paper \u003ca href=\"https://doi.org/10.1016/j.tiv.2022.105345\" rel=\"noreferrer noopener\" target=\"_blank\"\u003ehttps://doi.org/10.1016/j.tiv.2022.105345\u003c/a\u003e and available with the model code on Zenodo \u003ca href=\"https://zenodo.org/doi/10.5281/zenodo.5549494\" rel=\"noreferrer noopener\" target=\"_blank\"\u003e10.5281/zenodo.5549494\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cimg alt=\"\" src=\"https://aopwiki.org/system/dragonfly/production/2025/10/03/82u821si49_Fig_1_qAOP.jpg\" /\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eFigure 3: Predicted and observed KERs for rotenone for each readout (complex I activity, mitochondrial respiration, proteasomal activity, neurite area. Red line: predictions obtained with the maximum posterior vector. Grey lines: predictions obtained with 30 random parameter vectors drawn from their joint posterior distribution. Dots: observations of KEx at predicted KEx-1, colours represent replicates. (Reproduced from Tebby et al. 2022)\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cimg alt=\"\" src=\"https://aopwiki.org/system/dragonfly/production/2025/10/03/8qbyqv1gbz_Fig_4_qAOP.jpg\" /\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eFigure 4: Predicted and observed dose-response relationships for rotenone for each readout (complex I activity, mitochondrial respiration, proteasomal activity, neurite area. Red line: predictions obtained with the maximum posterior parameter values. Grey lines: predictions obtained with 30 random parameter vectors drawn from their joint posterior distribution. Dots: experimental data, colours represent replicates. (Reproduced from Tebby et al. 2022) \u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cimg alt=\"\" src=\"https://aopwiki.org/system/dragonfly/production/2025/10/03/3i370pf1o7_Fig_5_qAOP.jpg\" /\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eFigure 5: Predicted and observed KERs for deguelin for each readout (complex I activity, mitochondrial respiration, proteasomal activity, neurite area. Red line: predictions obtained with the maximum posterior vector. Grey lines: predictions obtained with 30 random parameter vectors drawn from their joint posterior distribution. Dots: observations of KEx at predicted KEx-1, colours represent replicates. (Reproduced from Tebby et al. 2022)\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cimg alt=\"\" src=\"https://aopwiki.org/system/dragonfly/production/2025/10/03/6vn63s5nbk_Fig_6_qAOP.jpg\" /\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eFigure 6: Predicted and observed dose-response relationships for deguelin for each readout (complex I activity, mitochondrial respiration, proteasomal activity, neurite area. Red line: predictions obtained with the maximum posterior parameter values. Grey lines: predictions obtained with 30 random parameter vectors drawn from their joint posterior distribution. Dots: experimental data, colours represent replicates. (Reproduced from Tebby et al. 2022) \u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eLimitations and uncertainties\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThis preliminary qAOP, based on a limited set of data, can be extended to include data identified in the literature and listed in section empirical evidence for cI inhibitors \u003ca href=\"https://aopwiki.org/relationships/934\" rel=\"noreferrer noopener\" target=\"_blank\"\u003eKER 934\u003c/a\u003e and \u003ca href=\"https://aopwiki.org/relationships/908\" rel=\"noreferrer noopener\" target=\"_blank\"\u003eKER 908\u003c/a\u003e (part of Revision of AOP3 Project:\u0026nbsp;\u003ca href=\"https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms\" rel=\"noreferrer noopener\" target=\"_blank\"\u003eNP/EFSA/PREV/2024/02\u003c/a\u003e), including for other stressors.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eData used to extend quantitative understanding was collected for a set of three compounds, rotenone, deguelin and tebufenpyrad, either by digitalizing summary data (mean and standard deviation) from publications, or by collecting data from the Biostudies online database. The data from Biostudies was visually compared to published data in order to establish correspondence between the datasets. Multiple-concentration data for cI inhibition (MIE/KE887) at 30 minutes was collected by digitalising figures from Alimohammadi et al. (2023) (tebufenpyrad) and from Biostudies S-TOX1365 (https://www.ebi.ac.uk/biostudies/eu-toxrisk/studies/S-TOXR1365) which could have been published in Tebby et al. (2022) (rotenone and deguelin). Multiple concentration data for OCR mitochondrial respiration (KE177) measured at 24 hours in LUHMES cells was collected from Alimohammadi et al. (2023) (tebufenpyrad) and from Biostudies S-TOXR1315 (https://www.ebi.ac.uk/biostudies/eu-toxrisk/studies/S-TOXR1315) which could have been published in Tebby et al. (2022) and van der Stel et al. (2020) (rotenone and deguelin). Multiple concentration data for ATP content (KE177) measured at 24 hours in LUHMES cells was collected from Biostudies S-TOXR1683 (https://www.ebi.ac.uk/biostudies/eu-toxrisk/studies/S-TOXR1683) which could have been published in Delp et al. (2021) (rotenone, \u0026nbsp;deguelin, tebufenpyrad). Multiple concentration data for cell viability and neurite area (KE890) measured at 24 hours in LUHMES cells was collected from Biostudies S-TOXR1203 (https://www.ebi.ac.uk/biostudies/eu-toxrisk/studies/S-TOXR1203) \u0026nbsp;which could have been published in Delp et al. (2021) (rotenone, \u0026nbsp;deguelin, tebufenpyrad), from Biostudies S-TOXR1279 (https://www.ebi.ac.uk/biostudies/eu-toxrisk/studies/S-TOXR1279) which could have been published in van der Stel et al. (2020) (rotenone, \u0026nbsp;deguelin) and from raw data provided by the authors of Alimohammadi et al. (2023) (tebufenpyrad).\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eBesides the uncertainty quantified by the statistical analysis including the individual replicate data, several sources of uncertainty are not accounted for in this preliminary qAOP.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eEach KE can be represented by several readouts; reassessment of the qAOP from Tebby et al. (2022) highlighted the following limitations regarding the choice of endpoints representing the KEs and regarding the KER between cI inhibition and the first KE.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eNeuron degeneration may not be adequately represented by neurite area measured in differentiating LUHMES cells during a 24-hour exposure starting a day 2 of differentiation. Repeated exposure in differentiated cells should be investigated. Furthermore, the KER between decrease in mitochondrial respiration and decrease in neurite area appeared to be substance specific when considering both rotenone and tebufenpyrad. A possible explanation is that effects of rotenone on microtubules could decrease neurite area at lower exposure concentrations than expected based on tebufenpyrad data and mitochondrial respiration data (recall the previously cited reference).\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eCell viability could be a relevant alternative to neurite area for representing neuron degeneration, however, the quantitative assessment revealed that the decrease in cell viability occurred at exposure concentrations at which mitochondrial respiration was already at its lowest level. Since both endpoints were measured in the same type of assay at the same timepoint, the exposure concentration levels between both endpoints are comparable. The lack of overlap between decrease of mitochondrial respiration and of cell viability suggests that other mechanisms are involved between these two key events or that the 24-hour exposure timepoint is not the most relevant.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eSeveral measurements of KE2 are possible: the decrease in ATP production could be an alternative endpoint to the decrease in mitochondrial respiration. Data on the resulting decrease in ATP content was available and was envisioned as a measurement of a downstream key event of mitochondrial respiration. However, decrease in mitochondrial respiration and in ATP content did not overlap in terms of effective concentration levels.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eThe lack of overlap in effective concentrations between decreases in mitochondrial respiration and ATP content or cell viability may be due to the ability of cells to switch from oxidative phosphorylation to glycolysis for energy production in the presence of glucose, compensating for mitochondrial inhibition. Assays using galactose as a substrate avoid this compensatory pathway. Nevertheless, dose-response data available in the literature had been mostly obtained in assays using glucose as a substrate rather than galactose. To enhance qAOP and lower KER uncertainty, more data in galactose conditions is needed to quantify the KERs using data obtained in LUHMES cells. Fortunately, the PANDORA project (OC/EFSA/PREV/2023/0, Environmental Neurotoxicants \u0026ndash; Advancing Understanding on the Impact of Chemical Exposure on Brain Health and Disease, LOT 3) is currently producing such data.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eEndpoints measured in the same experimental setup allow for comparisons of effective concentrations along the AOP. Complex I inhibition was measured in permeabilized cells, whereas the other endpoints that were quantitatively assessed were measured in intact cells. Intracellular exposure concentrations are therefore likely different and dependent on toxicokinetics. Chemical agnosticity of the KER between complex I inhibition and the decrease in mitochondrial respiration cannot be expected. The available data showed similar effective concentrations, but since the concentration levels in both assays may not be directly comparable due to differences in toxicokinetics, lack of overlap would not suggest a missing intermediate key event. Without intracellular concentration data, the comparison of effective concentrations in dissimilar assays provides only limited quantitative understanding of the KER.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eWhen exposure concentrations between adjacent KEs are comparable, quantitative AOP modelling would benefit from application of FAIR principles to data. Publication of raw data with metadata that could indicate which data were obtained with identical biological material and experimental conditions would help refine quantitative KERs. Data rich compounds such as rotenone sometimes showed large variations in potencies for a given endpoint.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eOverall, reassessment of the available data considering the preliminary qAOP developed by Tebby et al. (2022) highlighted several limitations inherent to the data used for qAOPs, in particular comparability of exposure concentrations between the various endpoints, comparability and relevance of timepoints and exposure durations, the necessary overlap in effective concentrations between adjacent endpoints that are measured at comparable exposure concentrations and relevant timepoints, and data reproducibility. Selection of most relevant readouts and accurate characterization of the molecular initiating event for cross-validation are critical when designing in vitro experiments targeted at calibrating qAOPs.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eAccording to the limits that were discussed, the following \u003cstrong\u003erecommendations\u003c/strong\u003e are made:\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eAs cultured cells rely predominantly on glycolysis as their primary source of ATP, it is recommended to replace glucose with galactose to shift cellular energy metabolism towards mitochondrial oxidative phosphorylation and increase cellular dependence on mitochondrial respiration.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eConsider a shorter exposure time of less than 24 hours for KE177 and increasing exposure duration over the 24h to identify potential effects on KE890\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eConsider using differentiated LUHMES cells rather than DoD2 to DoD3 to avoid the contribution of developmental pathways (e.g. neurite outgrowth) not any more active in a mature state.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eFuture developments of qAOP should include methods for modelling non overlapping adjacent Kes and research on the development and application\u0026nbsp;of biokinetic models for in vitro data that would accurately predict the internal concentration causing the effects.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e- Not \u003c/span\u003e\u003c/span\u003e\u003c/span\u003eendorsed\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\r\n","optional_considerations":"\u003col\u003e\r\n\t\u003cli\u003eThis AOP has been developed in order to evaluate the biological plausibility that the adverse outcome i.e. parkinsonian motor deficits, is linked to a MIE that can be triggered by chemical substances i.e. pesticides and chemicals in general. The relevance of the AOP has been documented by tools compounds known to trigger the described AOP. By means of using a human health outcome that has been shown in epidemiological studies to be association with pesticide exposure, the authors intend to draw attention on this AO in the process of hazard identification. This AOP can be used to support the biological plausibility of this association during the process of evaluation and integration of the epidemiological studies into the risk assessment. It is biologically plausible that a substance triggering the pathway, can be associated with the AO and ultimately with the human health outcome, pending the MoA analysis.\u003c/li\u003e\r\n\t\u003cli\u003eIn addition, this AOP can be used to support identification of data gaps that should be explored when a chemical substance is affecting the pathway. Moreover, the AOP provides a scaffold for recommendations on the most adequate study design to investigate the apical endpoints. It is important to note that, although the AO is defined in this AOP as parkinsonian motor deficits, degeneration of DA neurons is already per se an adverse outcome even in situations where it is not leading to parkinsonian motor deficits, and this should be taken into consideration for the regulatory applications of this AOP.\u003c/li\u003e\r\n\t\u003cli\u003eThe MIE and KEs identified in this AOP could serve as a basis for assays development that could contribute to an AOP informed-IATA construction which can be applied for different purposes such as: screening and prioritization of chemicals for further testing, hazard characterization or even risk assessment when combined with exposure and ADME information.\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003eThis AOP can be used for neurotoxicity assessment, since it is plausible that a compound that binds to the mitochondrial complex I may eventually lead to Parkinsonian motor deficits.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003eThe regulatory applicability of this AOP would be to use experimental findings in model systems representing the MIE and KEs as indicators/alerts for the AO. Risk assessment may be possible if bioavailability at the target cells can be estimated, the toxic concentrations \u003cem\u003ein vitro\u003c/em\u003e can be extrapolated to \u003cem\u003ein vivo\u003c/em\u003e and exposure scenarios can be simulated.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003eThis AOP can be applied for chemicals that have structural similarities to rotenone or MPTP. However, this AOP may not at the moment be used for chemicals that do not resemble rotenone or MPTP. It is however expected that compounds acting on the same MIE, but belonging to different chemical classes and those that are structurally different, will be tested in the near future in order to substantiate a broader specificity for this AOP. However, it remains evident that chemicals affecting the MIE are potential risk factors for this AO.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ol\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","references":"\u003cp\u003eAlimohammadi Mahshid, Birthe Meyburg, Anna-Katharina \u0026Uuml;ckert, Anna-Katharina Holzer,\u0026nbsp;Marcel Leist, 2023. EFSA Pilot Project on New Approach Methodologies (NAMs) for\u0026nbsp;Tebufenpyrad Risk Assessment. Part 2. Hazard characterisation and identification of the\u0026nbsp;Reference Point. EFSA supporting publication 2023:EN-7794. 56 pp.\u0026nbsp;doi:10.2903/sp.efsa.2023.EN-7794\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Abdelsalam%20RM%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=25752913\"\u003eAbdelsalam RM\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Safar%20MM%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=25752913\"\u003eSafar MM\u003c/a\u003e\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/25752913#\" title=\"Journal of neurochemistry.\"\u003eJ Neurochem.\u003c/a\u003e\u0026nbsp;\u003cspan style=\"font-size:14px\"\u003eNeuroprotective effects of vildagliptin in rat rotenone Parkinson\u0026#39;s disease model: role of RAGE-NF\u0026kappa;B and Nrf2-antioxidant signaling pathways.\u003c/span\u003e\u0026nbsp;2015 Jun;133(5):700-7. doi: 10.1111/jnc.13087. Epub 2015 Mar 26.\u003c/p\u003e\r\n\r\n\u003cp\u003eAguilar JS, Kostrzewa RM. Neurotoxin mechanisms and processes relevant to parkinson\u0026rsquo;s disease: un update. Neurotox Res. DOI 10.1007/s12640-015-9519-y.\u003c/p\u003e\r\n\r\n\u003cp\u003eAlvarez-Fischer D, Guerreiro S, Hunot S, Saurini F, Marien M, Sokoloff P, Hirsch EC, Hartmann A, Michel PP. Modelling Parkinson-like neurodegeneration via osmotic minipump delivery of MPTP and probenecid. J Neurochem. 2008 Nov;107(3):701-11. doi: 10.1111/j.1471-4159.2008.05651.x. Epub 2008 Sep 16.\u003c/p\u003e\r\n\r\n\u003cp\u003eArnold, B., et al. (2011). \u0026quot;Integrating Multiple Aspects of Mitochondrial Dynamics in Neurons: Age-Related Differences and Dynamic Changes in a Chronic Rotenone Model.\u0026quot; Neurobiology of Disease 41(1): 189-200.\u003c/p\u003e\r\n\r\n\u003cp\u003eBarbeito AG, Mesci P, Boillee S. 2010. Motor neuron-immune interactions: the vicious circle of ALS. J Neural Transm 117(8): 981-1000.\u003c/p\u003e\r\n\r\n\u003cp\u003eBalmaceda V, Koml\u0026oacute;di T, Szibor M, Gnaiger E, Moore AL, Fernandez-Vizarra E, Viscomi C. The striking differences in the bioenergetics of brain and liver mitochondria are enhanced in mitochondrial disease. Biochim Biophys Acta Mol Basis Dis. 2024 Mar;1870(3):167033. doi: 10.1016/j.bbadis.2024.167033. Epub 2024 Jan 26. PMID: 38280294.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eBarrientos A., and Moraes C.T. (1999) Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology. Vol. 274, No. 23, pp. 16188\u0026ndash;16197.\u003c/p\u003e\r\n\r\n\u003cp\u003eBernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F. Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurol Sci. 1973 Dec;20(4):415-55\u003c/p\u003e\r\n\r\n\u003cp\u003eBetarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. 2000. Chronic systemic pesticide exposure reproduces features of Parkinson\u0026rsquo;s disease. Nat Neurosci. 3:1301\u0026ndash;6\u003c/p\u003e\r\n\r\n\u003cp\u003eBetarbet R, Canet-Aviles RM, Sherer TB, Mastroberardino PG,Mc Lendon C, Kim JH, Lund S, Na HM, taylor G, Bence NF, kopito R, seo BB, Yagi T, Yagi A, Klinfelter G, Cookson MR, Greenmyre JT. 2006. Intersecting pathways to neurodegeneration in Parkinson\u0026rsquo;s disease: effects of the pesticide rotenone on DJ-1, \u0026alpha;-synuclein, and the ubiquitin-proteasome system. Neurobiology disease. (22) 404-20.\u003c/p\u003e\r\n\r\n\u003cp\u003eBezard E, Dovero S, Prunier C, Ravenscroft P, Chalon S, Guilloteau D, Crossman AR, Bioulac B, Brotchie JM, Gross CE (2001) Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson\u0026#39;s disease. J Neurosci. 21(17):6853-61.\u003c/p\u003e\r\n\r\n\u003cp\u003eBlasko I, Stampfer-Kountchev M, Robatscher P, Veerhuis R, Eikelenboom P, Grubeck-Loebenstein B. 2004. How chronic inflammation can affect the brain and support the development of Alzheimer\u0026#39;s disease in old age: the role of microglia and astrocytes. Aging cell 3(4): 169-176.\u003c/p\u003e\r\n\r\n\u003cp\u003eBlesa J, Pifl C, S\u0026aacute;nchez-Gonz\u0026aacute;lez MA, Juri C, Garc\u0026iacute;a-Cabezas MA, Ad\u0026aacute;nez R, Iglesias E, Collantes M, Pe\u0026ntilde;uelas I, S\u0026aacute;nchez-Hern\u0026aacute;ndez JJ, Rodr\u0026iacute;guez-Oroz MC, Avenda\u0026ntilde;o C, Hornykiewicz O, Cavada C, Obeso JA (2012) The nigrostriatal system in the presymptomatic and symptomatic stages in the MPTP monkey model: a PET, histological and biochemical study. Neurobiol Dis. 48(1):79-91.\u003c/p\u003e\r\n\r\n\u003cp\u003eBodea LG, Wang Y, Linnartz-Gerlach B, Kopatz J, Sinkkonen L, Musgrove R, et al. 2014. Neurodegeneration by activation of the microglial complement-phagosome pathway. J Neurosci 34(25): 8546-8556.\u003c/p\u003e\r\n\r\n\u003cp\u003eBorrajo A, Rodriguez-Perez AI, Villar-Cheda B, Guerra MJ, Labandeira-Garcia JL. 2014. Inhibition of the microglial response is essential for the neuroprotective effects of Rho-kinase inhibitors on MPTP-induced dopaminergic cell death. Neuropharmacology 85: 1-8\u003c/p\u003e\r\n\r\n\u003cp\u003eBrinkley BR, Barham SS, Barranco SC, and Fuller GM. 1974. Rotenone inhibition of spindle microtubule assembly in mammalian cells,\u0026rdquo; Experimental Cell Research. 85(1)41\u0026ndash;46.\u003c/p\u003e\r\n\r\n\u003cp\u003eBrown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355\u003c/p\u003e\r\n\r\n\u003cp\u003eBraun RJ. (2012). Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Front Oncol 2:182.\u003c/p\u003e\r\n\r\n\u003cp\u003eBrzozowski MJ, Jenner P, Rose S. 2015. Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP(+)-induced neuronal toxicity. J Neural Transm 122(6): 779-788.\u003c/p\u003e\r\n\r\n\u003cp\u003eBurr SP, Klimm F, Glynos A, Prater M, Sendon P, Nash P, Powell CA, Simard ML, Bonekamp NA, Charl J, Diaz H, Bozhilova LV, Nie Y, Zhang H, Frison M, Falkenberg M, Jones N, Minczuk M, Stewart JB, Chinnery PF. Cell lineage-specific mitochondrial resilience during mammalian organogenesis. Cell. 2023 Mar 16;186(6):1212-1229.e21. doi: 10.1016/j.cell.2023.01.034. Epub 2023 Feb 23. PMID: 36827974.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eCacquevel M, Lebeurrier N, Cheenne S, Vivien D. 2004. Cytokines in neuroinflammation and Alzheimer\u0026#39;s disease. Curr Drug Targets 5(6): 529-534.\u003c/p\u003e\r\n\r\n\u003cp\u003eCalne DB, Sandler M (1970) L-Dopa and Parkinsonism. Nature. 226(5240):21-4.\u003c/p\u003e\r\n\r\n\u003cp\u003eCannon JR, Tapias V, Na HM, Honick AS, Drolet RE, Greenamyre JT (2009) A highly reproducible rotenone model of Parkinson\u0026#39;s disease. Neurobiol Dis. 34(2):279-90.\u003c/p\u003e\r\n\r\n\u003cp\u003eCappelletti G, Maggioni MG, Maci R. 1999. Influence of MPP+ on the state of tubulin polymerisation in NGF-differentiated PC12 cells. J Neurosci Res. 56(1):28-35.\u003c/p\u003e\r\n\r\n\u003cp\u003eCao S, Theodore S, Standaert DG.2010. Fc gamma receprors are required for NF-kB signaling, microglial activation and dopaminergic neurodegeneration in an AAV-synuclein mouse model of Parkinson\u0026#39;s disease. molecular neurodegeneration.5-42.\u003c/p\u003e\r\n\r\n\u003cp\u003eCappelletti G, Pedrotti B, Maggioni MG, Maci R. 2001. Microtubule assembly is directly affected by MPP(+)in vitro. Cell Biol Int.25(10):981-4.\u003c/p\u003e\r\n\r\n\u003cp\u003eCastrioto A, Lozano AM, Poon YY, Lang AE, Fallis M, Moro E. 2011. Ten-year outcome of subthalamic stimulation in Parkinson disease: a blinded evaluation. Arch Neurol. 68(12):1550-6.\u003c/p\u003e\r\n\r\n\u003cp\u003eChang CY, Choi DK, Lee DK, Hong YJ, Park EJ. 2013. Resveratrol confers protection against rotenone-induced neurotoxicity by modulating myeloperoxidase levels in glial cells. PLoS One 8(4): e60654.\u003c/p\u003e\r\n\r\n\u003cp\u003eChampy et al. (2004). Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration in rats: possible relevance for atypical Parkinsonism in Guadeloupe. \u003cem\u003eJ Neurochem \u003c/em\u003e\u003cstrong\u003e88\u003c/strong\u003e: 63-69.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eChao YX, He BP, Tay SS. 2009. Mesenchymal stem cell transplantation attenuates blood brain barrier damage and neuroinflammation and protects dopaminergic neurons against MPTP toxicity in the substantia nigra in a model of Parkinson\u0026#39;s disease. J Neuroimmunol 216(1-2): 39-50.\u003c/p\u003e\r\n\r\n\u003cp\u003eChen Y, Zhang DQ, Liao Z, Wang B, Gong S, Wang C, Zhang MZ, Wang GH, Cai H, Liao FF, Xu JP 2015. Anti-oxidant polydatin (piceid) protects against substantia nigral motor degeneration in multiple rodent models of Parkinson\u0026#39;s disease. Mol Neurodegener. 2;10(1):4.\u003c/p\u003e\r\n\r\n\u003cp\u003eChinta SJ, Andersen JK (2006) Reversible inhibition of mitochondrial complex I activity following chronic dopaminergic glutathione depletion in vitro: implications for Parkinson\u0026#39;s disease. Free Radic Biol Med. 41(9):1442-8.\u003c/p\u003e\r\n\r\n\u003cp\u003eChoi WS., Kruse S.E., Palmiter R, Xia Z., (2008) Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP, or paraquat. PNAS, 105, 39, 15136-15141\u003c/p\u003e\r\n\r\n\u003cp\u003eChoi BS, Kim H, Lee HJ, Sapkota K, Park SE, Kim S, Kim SJ (2014) Celastrol from \u0026#39;Thunder God Vine\u0026#39; protects SH-SY5Y cells through the preservation of mitochondrial function and inhibition of p38 MAPK in a rotenone model of Parkinson\u0026#39;s disease. Neurochem Res. 39(1):84-96.\u003c/p\u003e\r\n\r\n\u003cp\u003eChiu CC, Yeh TH, Lai SC, Wu-Chou YH, Chen CH, Mochly-Rosen D, Huang YC, Chen YJ, Chen CL, Chang YM, Wang HL, Lu CS. 2015. Neuroprotective effects of aldehyde dehydrogenase 2 activation in rotenone-induced cellular and animal models of parkinsonism. Exp Neurol. 263:244-53.\u003c/p\u003e\r\n\r\n\u003cp\u003eChou AP, Li S, Fitzmaurice AG, Bronstein JM. 2010. Mechanisms of rotenone-induced proteasome inhibition. NeuroToxicology. 31:367\u0026ndash;372. Chung YC, Kim SR, Park JY, Chung ES, Park KW, Won SY, et al. 2011. Fluoxetine prevents MPTP-induced loss of dopaminergic neurons by inhibiting microglial activation. Neuropharmacology 60(6): 963-974.\u003c/p\u003e\r\n\r\n\u003cp\u003eCorreia SC, Santos RX, Perry G, Zhu X, Moreira PI, Smith MA. (2012). Mitochondrial importance in Alzheimer\u0026rsquo;s, Huntington\u0026rsquo;s and Parkinson\u0026rsquo;s diseases. Adv Exp Med Biol 724:205 \u0026ndash; 221.\u003c/p\u003e\r\n\r\n\u003cp\u003eCotzias GC, Papavasiliou PS, Gellene R. 1969. L-dopa in parkinson\u0026#39;s syndrome. N Engl J Med. 281(5):272.\u003c/p\u003e\r\n\r\n\u003cp\u003eCozzolino M, Ferri A, Valle C, Carri MT. (2013). Mitochondria and ALS: implications from novel genes and pathways. Mol Cell Neurosci 55:44 \u0026ndash; 49.\u003c/p\u003e\r\n\r\n\u003cp\u003eDagda RK, Banerjee TD and Janda E. 2013. How Parkinsonian Toxins Dysregulate the Autophagy Machinery. Int. J. Mol. Sci. 14:22163-22189.\u003c/p\u003e\r\n\r\n\u003cp\u003eDauer W, Kholodilov N, Vila M, Trillat AC, Goodchild R, Larsen KE, Staal R, Tieu K, Schmitz Y, Yuan CA, Rocha M, Jackson-Lewis V, Hersch S, Sulzer D, Przedborski S, Burke R, Hen R. 2002. Resistance of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP. Proc Natl Acad Sci U S A. 99(22):14524-9.\u003c/p\u003e\r\n\r\n\u003cp\u003eDauer W, Kholodilov N, Vila M, Trillat AC, Goodchild R, Larsen KE, Staal R, Tieu K, Schmitz Y, Yuan CA, Rocha M, Jackson-Lewis V, Hersch S, Sulzer D, Przedborski S, Burke R, Hen R. 2002. Resistance of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP. Proc Natl Acad Sci U S A. 99(22):14524-9.\u003c/p\u003e\r\n\r\n\u003cp\u003eDauer W, Przerdborski S. 2003. Parkinson\u0026rsquo;sdisease: Mechanisms and Models.Neuron. 39, 889-9.\u003c/p\u003e\r\n\r\n\u003cp\u003eDe Bie RM, de Haan RJ, Nijssen PC, Rutgers AW, Beute GN, Bosch DA, Haaxma R, Schmand B, Schuurman PR, Staal MJ, Speelman JD. 1999. Unilateral pallidotomy in Parkinson\u0026#39;s disease: a randomised, single-blind, multicentre trial. Lancet. 354(9191):1665-9.\u003c/p\u003e\r\n\r\n\u003cp\u003eDegli Esposti M, Ghelli A. 1994. The mechanism of proton and electron transport in mitochondrial complex I. Biochim Biophys Acta.1187(2):116\u0026ndash;120.\u003c/p\u003e\r\n\r\n\u003cp\u003eDehay B, Bove J, Rodriguez-Muela N, Perier C, Recasens A, Boya P, Vila M. 2010. Pathogenic lysosomal depletion in Parkinson\u0026rsquo;s disease. J. Neurosci. 30:12535\u0026ndash;12544.\u003c/p\u003e\r\n\r\n\u003cp\u003eDehmer T, Lindenau J, Haid S, Dichgans J, Schulz JB. 2000. Deficiency of inducible nitric oxide synthase protects against MPTP toxicity in vivo. J Neurochem 74(5): 2213-2216.\u003c/p\u003e\r\n\r\n\u003cp\u003eDehmelt, L and Shelley H. Neurite Outgrowth: A Flick of the Wrist. Current Biology, 2007 Volume 17, Issue 15, R611 \u0026ndash; R614\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eDelp J, Cediel-Ulloa A, Suciu I, Kranaster P, van Vugt-Lussenburg BM, Munic Kos V, van der Stel W, Carta G, Bennekou SH, Jennings P, van de Water B, Forsby A, Leist M. Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors. Arch Toxicol. 2021 Feb;95(2):591-615. doi: 10.1007/s00204-020-02970-5. Epub 2021 Jan 29. PMID: 33512557; PMCID: PMC7870626.\u003c/p\u003e\r\n\r\n\u003cp\u003eDelp J, Funke M, Rudolf F, Cediel A, Bennekou SH, van der Stel W, Carta G, Jennings P, Toma C, Gardner I, van de Water B, Forsby A, Leist M. Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants. Arch Toxicol. 2019 Jun;93(6):1585-1608. doi: 10.1007/s00204-019-02473-y. Epub 2019 Jun 12. PMID: 31190196. \u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eDeuschl G, Schade-Brittinger C, Krack P, Volkmann J, Sch\u0026auml;fer H, B\u0026ouml;tzel K, Daniels C, Deutschl\u0026auml;nder A, Dillmann U, Eisner W, Gruber D, Hamel W, Herzog J, Hilker R, Klebe S, Kloss M, Koy J, Krause M, Kupsch A, Lorenz D, Lorenzl S, Mehdorn HM, Moringlane JR, Oertel W, Pinsker MO, Reichmann H, Reuss A, Schneider GH, Schnitzler A, Steude U, Sturm V, Timmermann L, Tronnier V, Trottenberg T, Wojtecki L, Wolf E, Poewe W, Voges J; German Parkinson Study Group, Neurostimulation Section. 2006. A randomized trial of deep-brain stimulation for Parkinson\u0026#39;s disease. N Engl J Med. 355(9):896-908.\u003c/p\u003e\r\n\r\n\u003cp\u003eDexter D. T., Jenner P.. Parkinson disease: from pathology to molecular disease mechanisms. Free Radical Biology and Medicine 62 (2013) 132-144\u003c/p\u003e\r\n\r\n\u003cp\u003eDietz GPH, Stockhausen KV, Dietz B et al. (2008) Membrane-permeable Bcl-xL prevents MPTP-induced dopaminergic neuronal loss in the substantia nigra. J Neurochem 104:757-765. Doi:10.1111/j.1471-4159.2007.05028.\u003c/p\u003e\r\n\r\n\u003cp\u003eDrolet RE, Behrouz B, Lookingland KJ, Goudreau JL, 2004. Mice lacking \u0026alpha;-synuclein have an attenuated loss of striatal dopamine following prolonged chronic MPTP administration. Neurotoxicology. 25(5):761-9.\u003c/p\u003e\r\n\r\n\u003cp\u003eDrouin-Ouellet J, St-Amour I, Saint-Pierre M, Lamontagne-Prolux J, Kriz J, Barker R, Cicchetti F.2015. Toll-like receptor expression in the blood and brain of patients and a mouse of Parkinson\u0026#39;s disease. International Journal of Neuropsychopharmacology. 1-11.\u003c/p\u003e\r\n\r\n\u003cp\u003eDu T, Li L, Song N, Xie J, Jiang H (2010) Rosmarinic acid antagonized 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity in MES23.5 dopaminergic cells. Int J Toxicol. 29(6):625-33.\u003c/p\u003e\r\n\r\n\u003cp\u003eEfremov RG, Sazanov LA. Respiratory complex I: \u0026#39;steam engine\u0026#39; of the cell? Curr Opin Struct Biol. 2011 Aug;21(4):532-40. doi: 10.1016/j.sbi.2011.07.002. Epub 2011 Aug 8. Review.\u003c/p\u003e\r\n\r\n\u003cp\u003eEfremov RG, Sazanov LA. Structure of the membrane domain of respiratory complex I. Nature. 2011 Aug 7;476(7361):414-20. doi: 10.1038/nature10330.\u003c/p\u003e\r\n\r\n\u003cp\u003eEFSA Panel on Plant Protection Products and their residues (PPR); Ockleford C, Adriaanse P, Berny P, Brock T, Duquesne S, Grilli S, Hernandez-Jerez AF, Bennekou SH, Klein M, Kuhl T, Laskowski R, Machera K, Pelkonen O, Pieper S, Smith R, Stemmer M, Sundh I, Teodorovic I, Tiktak A, Topping CJ, Wolterink G, Angeli K, Fritsche E, Hernandez-Jerez AF, Leist M, Mantovani A, Menendez P, Pelkonen O, Price A, Viviani B, Chiusolo A, Ruffo F, Terron A, Bennekou SH. Investigation into experimental toxicological properties of plant protection products having a potential link to Parkinson\u0026#39;s disease and childhood leukaemia. EFSA J. 2017 Mar 16;15(3):e04691. doi: 10.2903/j.efsa.2017.4691. PMID: 32625422; PMCID: PMC7233269. \u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eEmmrich JV, Hornik TC, Neher JJ, Brown GC. 2013. Rotenone induces neuronal death by microglial phagocytosis of neurons. The FEBS journal 280(20): 5030-5038.\u003c/p\u003e\r\n\r\n\u003cp\u003eEsposti et al. (1993) Complex I and Complex III of mitochondria have common inhibitors acting as ubiquinone antagonists. \u003cem\u003eBiochem Biophys Res Commun \u003c/em\u003e\u003cstrong\u003e190\u003c/strong\u003e(3): 1090-6.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eFasano A, Romito LM, Daniele A, Piano C, Zinno M, Bentivoglio AR, Albanese A. 2010. Motor and cognitive outcome in patients with Parkinson\u0026#39;s disease 8 years after subthalamic implants. Brain. 133(9):2664-76.\u003c/p\u003e\r\n\r\n\u003cp\u003eFato et al. (2009) Differential effects of mitochondrial complex I inhibitors on production of reactive oxygen species. \u003cem\u003eBiochim Biophys Acta \u003c/em\u003e\u003cstrong\u003e1787\u003c/strong\u003e(5): 384-392.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eFeng ZH, Wang TG, Li DD, Fung P, Wilson BC, Liu B, et al. 2002. Cyclooxygenase-2-deficient mice are resistant to 1-methyl-4-phenyl1, 2, 3, 6-tetrahydropyridine-induced damage of dopaminergic neurons in the substantia nigra. Neurosci Lett 329(3): 354-358.\u003c/p\u003e\r\n\r\n\u003cp\u003eFeng J. Mictrotubule. A common target for parkin and Parkinson\u0026#39;s disease toxins. Neuroscientist 2006, 12.469-76.\u003c/p\u003e\r\n\r\n\u003cp\u003eFerger B, Leng A, Mura A, Hengerer B, Feldon J. 2004. Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNF-synthesis attenuates MPTP toxicity in mouse striatum. J Neurochem 89(4): 822-833.\u003c/p\u003e\r\n\r\n\u003cp\u003eFerrante RJ, Schulz JB, Kowall NW, Beal MF. 1997. Systematic administration of rotenone produces selective damage in the striatum and globus pallidus, but not in the substantia nigra. Brain Research. (753). 157-2.\u003c/p\u003e\r\n\r\n\u003cp\u003eFerrari-Toninelli G, Bonini SA, Cenini G, Maccarinelli G, Grilli M, Uberti D, Memo M. 2008. Dopamine receptor agonists for protection and repair in Parkinson\u0026#39;s disease. Curr Top Med Chem. 8(12):1089-99.\u003c/p\u003e\r\n\r\n\u003cp\u003eFriedman LG, lachenmayer ML, Wang J, He L, Poulose SM, Komatsu M, Holstein GR, Yue Z. 2012. Disrupted autophagy leads to dopaminergic axon and dendrite degeneration and promotes presynaptic accumulation of \u0026alpha;-synuclein and LRRK2 in the brain. The Journal of Neuroscience. 32 (22) 7585-93.\u003c/p\u003e\r\n\r\n\u003cp\u003eFornai F, Lenzi P, Gesi M, Ferrucci M, Lazzeri G, Busceti C, Ruffoli R, Soldani P, Ruggieri S, Alessandri\u0026rsquo; MG, Paparelli A. 2003. Fine structure and mechanisms underlying nigrostriatal inclusions and cell death after proteasome inhibition. The journal of neuroscience. 23 (26) 8955-6.\u003c/p\u003e\r\n\r\n\u003cp\u003eFornai F., P. Lenzi, M. Gesi et al., \u0026ldquo;Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells,\u0026rdquo; Journal of Neurochemistry, vol. 88, no. 1, pp. 114\u0026ndash;123, 2004.\u003c/p\u003e\r\n\r\n\u003cp\u003eFornai F, Schl\u0026uuml;ter OM, Lenzi P, Gesi M, Ruffoli R, Ferrucci M, Lazzeri G, Busceti CL, Pontarelli F, Battaglia G, Pellegrini A, Nicoletti F, Ruggieri S, Paparelli A, S\u0026uuml;dhof TC. 2005. Parkinson-like syndrome induced by continuous MPTP infusion: Convergent roles of the ubiquitinproteasome system and _\u0026alpha;-synuclein. PNAS. 102: 3413\u0026ndash;3418.\u003c/p\u003e\r\n\r\n\u003cp\u003eFreed CR, Breeze RE, Rosenberg NL, Schneck SA, Wells TH, Barrett JN, Grafton ST, Huang SC, Eidelberg D, Rottenberg DA. 1990.\u003c/p\u003e\r\n\r\n\u003cp\u003eTransplantation of human fetal dopamine cells for Parkinson\u0026#39;s disease. Results at 1 year. Arch Neurol. 47(5):505-12.\u003c/p\u003e\r\n\r\n\u003cp\u003eFujita KA, Ostaszewski M, Matsuoka Y, Ghosh S, Glaab E, Trefois C, Crespo I, Perumal TM, Jurkowski W, Antony PM, Diederich N, Buttini M, Kodama A, Satagopam VP, Eifes S, Del Sol A, Schneider R, Kitano H, Balling R. 2014. Integrating pathways of Parkinson\u0026#39;s disease in a molecular interaction map. Mol Neurobiol.49(1):88-102.\u003c/p\u003e\r\n\r\n\u003cp\u003eGandhi S, Wood-Kaczmar A, Yao Z, et al. PINK1-associated Parkinson\u0026rsquo;s disease is caused by neuronal vulnerability to calcium-induced cell death. Molecular Cell. 2009;33:627\u0026ndash;638.\u003c/p\u003e\r\n\r\n\u003cp\u003eGao HM, Hong JS, Zhang W, Liu B. 2002. Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. J Neurosci 22(3): 782-790.\u003c/p\u003e\r\n\r\n\u003cp\u003eGao HM, Liu B, Hong JS. 2003. Critical role for microglial NADPH oxidase in rotenone-induced degeneration of dopaminergic neurons. J Neurosci 23(15): 6181-6187.\u003c/p\u003e\r\n\r\n\u003cp\u003eGao L, Brenner D, Llorens-Bobadilla E, Saiz-Castro G, Frank T, Wieghofer P, et al. 2015. Infiltration of circulating myeloid cells through CD95L contributes to neurodegeneration in mice. J Exp Med 212(4): 469-480.\u003c/p\u003e\r\n\r\n\u003cp\u003eGonz\u0026aacute;lez-Rodr\u0026iacute;guez, P., Zampese, E., Stout, K.A. et al. Disruption of mitochondrial complex I induces progressive parkinsonism. Nature 599, 650\u0026ndash;656 (2021). https://doi.org/10.1038/s41586-021-04059-0\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eGraier WF, Frieden M, Malli R. (2007). Mitochondria and Ca2+ signaling: old guests, new functions. Pflugers Arch 455:375\u0026ndash;396.\u003c/p\u003e\r\n\r\n\u003cp\u003eGreenamyre, J T., Sherer, T.B., Betarbet, R., and Panov A.V. (2001) Critical Review Complex I and Parkinson\u0026rsquo;s Disease Life, 52: 135\u0026ndash;141.\u003c/p\u003e\r\n\r\n\u003cp\u003eGreenamayre et al. 2010. Lessons from the rotenone model of Parkinson\u0026#39;s disease. Trends pharmacol. Sci. 31(4):141-2\u003c/p\u003e\r\n\r\n\u003cp\u003eGriffin WS, Sheng JG, Royston MC, Gentleman SM, McKenzie JE, Graham DI, et al. 1998. Glial-neuronal interactions in Alzheimer\u0026#39;s disease: the potential role of a \u0026#39;cytokine cycle\u0026#39; in disease progression. Brain Pathol 8(1): 65-72.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nGrivennikova, V.G., Maklashina, E.O., E.V. Gavrikova, A.D. Vinogradov (1997) Interaction of the mitochondrial NADH-ubiquinone reductase with rotenone as related to the enzyme active/inactive transition Biochim. Biophys. Acta, 1319 (1997), pp. 223\u0026ndash;232\u003c/p\u003e\r\n\r\n\u003cp\u003eGusdon AM, Fernandez-Bueno GA, Wohlgemuth S, Fernandez J, Chen J, Mathews CE. Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver. BMC Biochem. 2015 Sep 10;16:22. doi: 10.1186/s12858-015-0051-8. PMID: 26358560; PMCID: PMC4564979.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eHajieva P, Mocko JB, Moosmann B, Behl C (2009) Novel imine antioxidants at low nanomolar concentrations protect dopaminergic cells from oxidative neurotoxicity. J Neurochem. 110(1):118-32.\u003c/p\u003e\r\n\r\n\u003cp\u003eHoglinger G.U. et al.2003.Chronic systemic complex I inhibition induces a hypokynetic multisystem degeneration in rats. J.neurochem 84:491-502.\u003c/p\u003e\r\n\r\n\u003cp\u003eHornykiewicz O, Kish SJ. 1987. Biochemical pathophysiology of parkinson\u0026rsquo;s disease. In Parkinson\u0026rsquo;s Disease. M Yahr and K.J. Bergmann, eds (New.York: Raven Press) 19-34.\u003c/p\u003e\r\n\r\n\u003cp\u003eJana S, Sinha M, Chanda D, Roy T, Banerjee K, Munshi S, Patro BS, Chakrabarti S (2011) Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson\u0026#39;s disease. Biochim Biophys Acta. 1812(6):663-73.\u003c/p\u003e\r\n\r\n\u003cp\u003eJha N, Jurma O, Lalli G, Liu Y, Pettus EH, Greenamyre JT, Liu RM, Forman HJ, Andersen JK (2000) Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson\u0026#39;s disease. J Biol Chem. 275(34):26096-101.\u003c/p\u003e\r\n\r\n\u003cp\u003eJohnson ME, Bobrovskaya L. 2015. An update on the rotenone models of parkinson\u0026rsquo;s disease: Their ability to reproduce features of clinical disease and model gene-environment interactions. 946). 101-16.\u003c/p\u003e\r\n\r\n\u003cp\u003eHeikkila RE, Nicklas WJ, Vyas I, Duvoisin RC. 1985. Dopaminergic toxicity of rotenone and the 1-methyl-4-phenylpyridinium ion after their stereotaxic administration to rats: implication for the mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity. Neurosci Lett. 62(3):389-94.\u003c/p\u003e\r\n\r\n\u003cp\u003eHenderson BT, Clough CG, Hughes RC, Hitchcock ER, Kenny BG. 1991. Implantation of human fetal ventral mesencephalon to the right caudate nucleus in advanced Parkinson\u0026#39;s disease. Arch Neurol. 48(8):822-7.\u003c/p\u003e\r\n\r\n\u003cp\u003eHirsch EC, Hunot S. 2009. Neuroinflammation in Parkinson\u0026#39;s disease: a target for neuroprotection? Lancet Neurol 8(4): 382-397. Hoglinger GU, Feger J, Annick P, Michel PP, Karine P, Champy P, Ruberg M, Wolfgang WO, Hirsch E. 2003. Chronic systemic complexI inhibition induces a hypokinetic multisystem degeneration in rats. J. Neurochem.. (84) 1-12.\u003c/p\u003e\r\n\r\n\u003cp\u003eIchimaru N, Murai M, Kakutani N, Kako J, Ishihara A, Nakagawa Y, Miyoshi H. 2008.. Synthesis and Characterization of New Piperazine-Type Inhibitors for Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I). Biochemistry. 47(40)10816\u0026ndash;10826.\u003c/p\u003e\r\n\r\n\u003cp\u003eInden M, Yoshihisa Kitamura, Hiroki Takeuchi, Takashi Yanagida, Kazuyuki Takata, Yuka Kobayashi, Takashi Taniguchi, Kanji Yoshimoto, Masahiko Kaneko, Yasunobu Okuma, Takahiro Taira, Hiroyoshi Ariga and Shun Shimohama. 2007. Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone. Journal of Neurochemistry. 101.(6).1491\u0026ndash;4.\u003c/p\u003e\r\n\r\n\u003cp\u003eKeeney PM,Xie J,Capaldi RA,Bennett JP Jr. (2006) Parkinson\u0026#39;s disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci. 10;26(19):5256-64.\u003c/p\u003e\r\n\r\n\u003cp\u003eKelly PJ, Ahlskog JE, Goerss SJ, Daube JR, Duffy JR, Kall BA. 1987. Computer-assisted stereotactic ventralis lateralis thalamotomy with microelectrode recording control in patients with Parkinson\u0026#39;s disease. Mayo Clin Proc. 62(8):655-64.\u003c/p\u003e\r\n\r\n\u003cp\u003eKhan MM, Kempuraj D, Zaheer S, Zaheer A. 2014. Glia maturation factor deficiency suppresses 1-methyl-4-phenylpyridinium-induced oxidative stress in astrocytes. J Mol Neurosci 53(4): 590-599.\u003c/p\u003e\r\n\r\n\u003cp\u003eKim-Han JS, Dorsey JA, O\u0026rsquo;Malley KL. 2011. The parkinsonian mimetic MPP+, specifically impairs mitochondrial transport in dopamine axons. The Journal of Neuroscience. 31(19) 7212-1.\u003c/p\u003e\r\n\r\n\u003cp\u003eKirk D, Rosenblad C, Burger C, Lundberg C, Johansen TE, Muzyczka N, Mandel R, Bijorklund A. 2002. Parkinson-like neurodegeneration induced by targeted overexpression of \u0026alpha;-synuclein in the nigrostriatal system. 22(7) 2780-91.\u003c/p\u003e\r\n\r\n\u003cp\u003eKirk D, Annett L, Burger C, Muzyczka N, Mandel R, Bijorklund A. 2003. Nigrostriatal \u0026alpha;-synucleinopathy induced by viral vector-mediated overexpression of human \u0026alpha;-synuclein: A new primate model of parkinson\u0026rsquo;s disease. PNAS (100) 2884-9.\u003c/p\u003e\r\n\r\n\u003cp\u003eKlein RL, King MA, Hamby ME, Meyer EM. 2002. Dopaminergic cell loss induced by human A30P \u0026alpha;-synuclein gene transfer to the rat substantia nigra. Hum.Gene.Ther. (13) 605-2.\u003c/p\u003e\r\n\r\n\u003cp\u003eKoller WC (1992) When does Parkinson\u0026#39;s disease begin? Neurology. 42(4 Suppl 4):27-31 Koopman W, Hink M, Verkaart S, Visch H, Smeitink J, Willems P. 2007. Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy. Biochimica et Biophysica Acta 1767:940-947.\u003c/p\u003e\r\n\r\n\u003cp\u003eKoopman W, Willems P (2012) Monogenic mitochondrial disorders. New Engl J Med. 22;366(12):1132-41. doi: 10.1056/NEJMra1012478.\u003c/p\u003e\r\n\r\n\u003cp\u003eKraft AD, Harry GJ. 2011. Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity. International journal of environmental research and public health 8(7): 2980-3018.\u003c/p\u003e\r\n\r\n\u003cp\u003eLagoa et al. (2011) Complex I and cytochrome \u003cem\u003ec \u003c/em\u003eare molecular targets of flavonoids that inhibit hydrogen peroxide production by mitochondria. \u003cem\u003eBiochimica et Biophys Acta\u003c/em\u003e \u003cstrong\u003e1807\u003c/strong\u003e: 1562-1572.\u003c/p\u003e\r\n\r\n\u003cp\u003eLang AE, Lozano AM. 1998. Parkinson\u0026#39;s disease. Second of two parts. N Engl J Med. 339(16):1130-43.\u003c/p\u003e\r\n\r\n\u003cp\u003eLangston JW, ballard P, Irwin I. 1983. Chronic parkinsonism in human due to a product of meperidine-analog synthesis. Science. (219) 979-0.\u003c/p\u003e\r\n\r\n\u003cp\u003eLapointe N, StHilaire M, martinoli MG, Blanchet J, gould P, Rouillard C, Cicchetti F. 2004. Rotenone induces non-specific central nervous system and systemic toxicity. The FASEB Journal express article 10.1096/fj.03-0677fje\u003c/p\u003e\r\n\r\n\u003cp\u003eLauwers E, Debyser Z, Van Drope J, DeStrooper B, Nuttin B. 2003. Neuropathology and neurodegeneration in rodent brain induced by lentiviral vector-mediated overexpression of \u0026alpha;-synuclein. Brain pathol. (13) 364-72.\u003c/p\u003e\r\n\r\n\u003cp\u003eLesner, N. P., Wang, X., Chen, Z., Frank, A., Menezes, C. J., House, S., ... \u0026amp; Mishra, P. (2022). Differential requirements for mitochondrial electron transport chain components in the adult murine liver. Elife, 11, e80919.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eLiu Y, Hu J, Wu J, Zhu C, Hui Y, Han Y, et al. 2012. alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation. J Neuroinflammation 9: 98.\u003c/p\u003e\r\n\r\n\u003cp\u003eLiu Y, Li W, Tan C, Liu X, Wang X, Gui Y, Qin L, Deng F, Hu C, Chen L. 2014. Meta-analysis comparing deep brain stimulation of the globus pallidus and subthalamic nucleus to treat advanced Parkinson disease. J Neurosurg. 121(3):709-18.\u003c/p\u003e\r\n\r\n\u003cp\u003eLiu Y, Zeng X, Hui Y, Zhu C, Wu J, Taylor DH, et al. 2015. Activation of alpha7 nicotinic acetylcholine receptors protects astrocytes against oxidative stress-induced apoptosis: implications for Parkinson\u0026#39;s disease. Neuropharmacology 91: 87-96.\u003c/p\u003e\r\n\r\n\u003cp\u003eLiu W, Kong S, Xie Q, Su J, Li W, Guo H, Li S, Feng X, Su Z, Xu Y, Lai X. Protective effects of apigenin against 1-methyl-4-phenylpyridinium ion induced neurotoxicity in PC12 cells. Int J Mol Med. 2015, 35(3):739-46.\u003c/p\u003e\r\n\r\n\u003cp\u003eLloyd KG, Davidson L, Hornykiewicz O (1975) The neurochemistry of Parkinson\u0026#39;s disease: effect of L-dopa therapy. J Pharmacol Exp Ther. 195(3):453-64.\u003c/p\u003e\r\n\r\n\u003cp\u003eLo Bianco C, Ridet JL, Deglon N, Aebischer P. 2002. Alpha-synucleopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson\u0026rsquo;s disease. Proc.natl.Sci.USA (99)10813-8.\u003c/p\u003e\r\n\r\n\u003cp\u003eL\u0026oacute;pez-Lozano JJ, Bravo G, Abascal J. 1991. Grafting of perfused adrenal medullary tissue into the caudate nucleus of patients with Parkinson\u0026#39;s disease. Clinica Puerta de Hierro Neural Transplantation Group. J Neurosurg. 75(2):234-43.\u003c/p\u003e\r\n\r\n\u003cp\u003eMangano EN, Litteljohn D, So R, Nelson E, Peters S, Bethune C, et al. 2012. Interferon-gamma plays a role in paraquat-induced neurodegeneration involving oxidative and proinflammatory pathways. Neurobiol Aging 33(7): 1411-1426.\u003c/p\u003e\r\n\r\n\u003cp\u003eMarella M, Seo BB, Nakamaru-Ogiso E, Greenamyre JT, Matsuno-Yagi A, Yagi T (2008) Protection by the NDI1 gene against neurodegeneration in a rotenone rat model of Parkinson\u0026#39;s disease. PLoS One. 3(1):e1433.\u003c/p\u003e\r\n\r\n\u003cp\u003eMarshall, L. E. \u0026amp; Himes, R. H. Rotenone inhibition of tubulin self-assembly. Biochim Biophys Acta 543, 590\u0026ndash;594 (1978).\u003c/p\u003e\r\n\r\n\u003cp\u003eMartin LJ. (2011). Mitochondrial pathobiology in ALS. J Bioenerg Biomembr 43:569 \u0026ndash; 579.\u003c/p\u003e\r\n\r\n\u003cp\u003eMatsumoto K, Asano T, Baba T, Miyamoto T, Ohmoto T. 1976. Long-term follow-up results of bilateral thalamotomy for parkinsonism. Appl Neurophysiol. 39(3-4):257-60.\u003c/p\u003e\r\n\r\n\u003cp\u003eMcGeer PL, McGeer EG. 1998. Glial cell reactions in neurodegenerative diseases: Pathophysiology and therapeutic interventions. Alzheimer DisAssocDisord 12 Suppl. 2: S1-S6.\u003c/p\u003e\r\n\r\n\u003cp\u003eMcNaught KS, Jenner P. 2001. Proteasomal function is impaired in substantia nigra in Parkinson\u0026rsquo;s disease. Neurosci. Lett. 297, 191\u0026ndash; 194. McNaught KSC, Olanow W, Halliwell B. 2001. Failure of the ubiquitine-proteasome system in parkinson\u0026rsquo;s disease. Nature Rev. Neurosci. (2) 589-4.\u003c/p\u003e\r\n\r\n\u003cp\u003eMcNaught KS, Belizaire R, Isacson O, Jenner P, Olanow CW. 2003. Altered proteasomal function in sporadic Parkinson\u0026rsquo;s disease. Exp. Neurol. 179, 38\u0026ndash; 46.\u003c/p\u003e\r\n\r\n\u003cp\u003eMoldovan AS, Groiss SJ, Elben S, S\u0026uuml;dmeyer M, Schnitzler A, Wojtecki L. 2015. The treatment of Parkinson\u0026#39;s disease with deep brain stimulation: current issues. Neural Regen Res. 10(7):1018-22.\u003c/p\u003e\r\n\r\n\u003cp\u003eMud\u0026ograve; G, M\u0026auml;kel\u0026auml; J, Di Liberto V, Tselykh TV, Olivieri M, Piepponen P, Eriksson O, M\u0026auml;lki\u0026auml; A, Bonomo A, Kairisalo M, Aguirre JA, Korhonen L, Belluardo N, Lindholm D. (2012) Transgenic expression and activation of PGC-1\u0026alpha; protect dopaminergic neurons in the MPTP mouse model of Parkinson\u0026#39;s disease. Cell Mol Life Sci. 69(7):1153-65.\u003c/p\u003e\r\n\r\n\u003cp\u003eNarabayashi H, Yokochi F, Nakajima Y. 1984. Levodopa-induced dyskinesia and thalamotomy. J Neurol Neurosurg Psychiatry. 47(8):831-9. Nataraj J, Manivasagam T, Justin Thenmozhi A, Essa MM 2015. Lutein protects dopaminergic neurons against MPTP-induced apoptotic death and motor dysfunction by ameliorating mitochondrial disruption and oxidative stress. Nutr Neurosci. 2015 Mar 2. [Epub ahead of print].\u003c/p\u003e\r\n\r\n\u003cp\u003eObeso JA, Rodr\u0026iacute;guez-Oroz MC, Rodr\u0026iacute;guez M, Lanciego JL, Artieda J, Gonzalo N, Olanow CW (2000) Pathophysiology of the basal ganglia in Parkinson\u0026#39;s disease. Trends Neurosci. 23(10 Suppl):S8-19.\u003c/p\u003e\r\n\r\n\u003cp\u003eOffen D, Beart PM, Cheung NS et al. (1998) Transegnic mice expressing human Bcl-2 in their neurons are resistant to 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine neurotoxicity. PNAS 95:5789-5794\u003c/p\u003e\r\n\r\n\u003cp\u003eO\u0026rsquo;Malley KL. 2010. The role of axonopathy in Parkinson\u0026rsquo;s disease. 2010. Experimental Neurobiology. (19). 115-19.\u003c/p\u003e\r\n\r\n\u003cp\u003eOkun, J.G, L\u0026uuml;mmen, P and Brandt U., (1999) Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase) J. Biol. Chem. 274: 2625-2630. doi:10.1074/jbc.274.5.2625\u003c/p\u003e\r\n\r\n\u003cp\u003ePan T, Kondo S, Le W, Jankovic J. 2008. The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson\u0026rsquo;s disease. Brain. 131, 1969-1978.\u003c/p\u003e\r\n\r\n\u003cp\u003ePark C.et al. (2003) Quercetin protects the hydrogen peroxide-induced apoptosis via inhibition of mitochondrial dysfunction in H9c2 cardiomyoblast cells. \u003cem\u003eBiochem Pharmacol \u003c/em\u003e\u003cstrong\u003e66\u003c/strong\u003e(7): 1287-1295.\u003c/p\u003e\r\n\r\n\u003cp\u003ePark SE, Sapkota K, Choi JH, Kim MK, Kim YH, Kim KM, Kim KJ, Oh HN, Kim SJ, Kim S (2014) Rutin from Dendropanax morbifera Leveille protects human dopaminergic cells against rotenone induced cell injury through inhibiting JNK and p38 MAPK signaling. Neurochem Res. 39(4):707-18.\u003c/p\u003e\r\n\r\n\u003cp\u003eParker WD Jr, Boyson SJ, Parks JK. 1989. Abnormalities of the electron transport chain in idiopathic Parkinson\u0026#39;s disease. Ann Neurol.26(6):719-23.\u003c/p\u003e\r\n\r\n\u003cp\u003epasqualiL, Caldarazzo-Ienco Fornai . (2014). MPTP neurotoxicity:actions, mechanisms, and animal modeling of Parkinson\u0026#39;s disease. In: Kostrzewa RM (ed) Handbook of neurotoxicity. Springer, Heidelberg, pp237-275.\u003c/p\u003e\r\n\r\n\u003cp\u003ePeschanski M, Defer G, N\u0026#39;Guyen JP, Ricolfi F, Monfort JC, Remy P, Geny C, Samson Y, Hantraye P, Jeny R. 1994. Bilateral motor improvement and alteration of L-dopa effect in two patients with Parkinson\u0026#39;s disease following intrastriatal transplantation of foetal ventral mesencephalon. Brain. 117 ( Pt 3):487-99.\u003c/p\u003e\r\n\r\n\u003cp\u003ePetroske E, Meredith GE, Callen S, Totterdell S, Lau YS (2001) Mouse model of Parkinsonism: a comparison between subacute MPTP and chronic MPTP/probenecid treatment. Neuroscience. 106(3):589-601.\u003c/p\u003e\r\n\r\n\u003cp\u003ePowers ET1, Morimoto RI, Dillin A, Kelly JW, Balch WE. 2009.. Biological and Chemical Approaches to Diseases of Proteostasis Deficiency. Ann. Rev. Biochem 78: 959\u0026ndash;91.\u003c/p\u003e\r\n\r\n\u003cp\u003ePurisai MG, McCormack AL, Cumine S, Li J, Isla MZ, Di Monte DA. 2007. Microglial activation as a priming event leading to paraquat-induced dopaminergic cell degeneration. Neurobiol Dis 25(2): 392-400. Parker WD Jr, Parks JK, Swerdlow RH (2008) Complex I deficiency in Parkinson\u0026#39;s disease frontal cortex. Brain Res. 1189:215-8.\u003c/p\u003e\r\n\r\n\u003cp\u003eQian L, Wu HM, Chen SH, Zhang D, Ali SF, Peterson L, et al. 2011. beta2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway. J Immunol 186(7): 4443-4454.\u003c/p\u003e\r\n\r\n\u003cp\u003eRappold PM et al.2014. Drp1 inhibition attenuates neurotoxicity and dopamine release deficits in vivo. Nature Communications. 5:5244 doi: 10.1038/ncomms6244.\u003c/p\u003e\r\n\r\n\u003cp\u003eRen Y. et al., 2005. Selectivwe vulnerabity of dopaminergic neurons to microtubule depolymerisation. J. Bio. Chem. 280:434105-12. Reynolds GP, Garrett NJ (1986) Striatal dopamine and homovanillic acid in Huntington\u0026#39;s disease. J Neural Transm. 65(2):151-5.\u003c/p\u003e\r\n\r\n\u003cp\u003eRiederer BM, Pellier V, Antonsson B, Di Paolo G, Stimpson SA, L\u0026uuml;tjens R, Catsicas S, Grenningloh G. Regulation of microtubule dynamics by the neuronal growth-associated protein SCG10. Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):741-5. doi: 10.1073/pnas.94.2.741. PMID: 9012855; PMCID: PMC19584.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eRojo AI, Innamorato NG, Martin-Moreno AM, De Ceballos ML, Yamamoto M, Cuadrado A. 2010. Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson\u0026#39;s disease. Glia 58(5): 588-598.\u003c/p\u003e\r\n\r\n\u003cp\u003eRos-Bernal F, Hunot S, Herrero MT, Parnadeau S, Corvol JC, Lu L, et al. 2011. Microglial glucocorticoid receptors play a pivotal role in regulating dopaminergic neurodegeneration in parkinsonism. Proc Natl Acad Sci U S A 108(16): 6632-6637.\u003c/p\u003e\r\n\r\n\u003cp\u003eRossignol R, Malgat M, Mazat JP, Letellier T. Threshold effect and tissue specificity. Implication for mitochondrial cytopathies. J Biol Chem. 1999 Nov 19;274(47):33426-32. doi: 10.1074/jbc.274.47.33426. PMID: 10559224.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eRossignol R, Letellier T, Malgat M, Rocher C, Mazat JP. Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases. Biochem J. 2000 Apr 1;347 Pt 1(Pt 1):45-53. PMID: 10727400; PMCID: PMC1220929.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eRubio-Perez JM, Morillas-Ruiz JM. 2012. A review: inflammatory process in Alzheimer\u0026#39;s disease, role of cytokines. ScientificWorldJournal 2012: 756357.\u003c/p\u003e\r\n\r\n\u003cp\u003eSalama M, Helmy B, El-Gamal M, Reda A, Ellaithy A, Tantawy D, et al. 2013. Role of L-thyroxin in counteracting rotenone induced neurotoxicity in rats. Environmental toxicology and pharmacology 35(2): 270-277.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Sandstr%C3%B6m%20J%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=28467894\"\u003eSandstr\u0026ouml;m J\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Broyer%20A%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=28467894\"\u003eBroyer A\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Zoia%20D%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=28467894\"\u003eZoia D\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Schilt%20C%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=28467894\"\u003eSchilt C\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Greggio%20C%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=28467894\"\u003eGreggio C\u003c/a\u003e1,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Fournier%20M%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=28467894\"\u003eFournier M\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Do%20KQ%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=28467894\"\u003eDo KQ\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Monnet-Tschudi%20F%5BAuthor%5D\u0026amp;cauthor=true\u0026amp;cauthor_uid=28467894\"\u003eMonnet-Tschudi F\u003c/a\u003e.\u0026nbsp;\u003cspan style=\"font-size:12px\"\u003ePotential mechanisms of development-dependent adverse effects of the herbicide paraquat in 3D rat brain cell cultures.\u003c/span\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/28467894#\" title=\"Neurotoxicology.\"\u003eNeurotoxicology.\u003c/a\u003e\u0026nbsp;2017 May;60:116-124. doi: 10.1016/j.neuro.2017.04.010. Epub 2017 Apr 30.\u003c/p\u003e\r\n\r\n\u003cp\u003eSaravanan KS, Sindhu KM, Senthilkumar KS, Mohanakumar KP. 2006. L-deprenyl protects against rotenone-induced, oxidative stress-mediated dopaminergic neurodegeneration in rats. Neurochem Int.49(1):28-40.\u003c/p\u003e\r\n\r\n\u003cp\u003eSathe K, Maetzler W, Lang JD, Mounsey RB, Fleckenstein C, Martin HL, et al. 2012. S100B is increased in Parkinson\u0026#39;s disease and ablation protects against MPTP-induced toxicity through the RAGE and TNF-alpha pathway. Brain 135(Pt 11): 3336-3347.\u003c/p\u003e\r\n\r\n\u003cp\u003eSchapira AH, Cooper JM, Dexter D, Jenner P, Clark JB, and Marsden CD. 1989. Mitochondrial complex I de. ciency in Parkinson\u0026rsquo;s disease. Lancet. 1,1269.\u003c/p\u003e\r\n\r\n\u003cp\u003eScott R, Gregory R, Hines N, Carroll C, Hyman N, Papanasstasiou V, Leather C, Rowe J, Silburn P, Aziz T. 1998. Neuropsychological, neurological and functional outcome following pallidotomy for Parkinson\u0026#39;s disease. A consecutive series of eight simultaneous bilateral and twelve unilateral procedures. Brain. 121 ( Pt 4):659-75.\u003c/p\u003e\r\n\r\n\u003cp\u003eSherer TB, Betarbet R, Stout AK, Lund S, Baptista M, Panov AV, Cookson MR, Greenamyre JT. 2002. An in vitro model of Parkinson\u0026#39;s disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage. J Neurosci. 22(16):7006-15.\u003c/p\u003e\r\n\r\n\u003cp\u003eSherer TB, Betarbet R, Testa CM, Seo BB, Richardson JR, Kim JH, et al. 2003. Mechanism of toxicity in rotenone models of Parkinson\u0026rsquo;s disease. J Neurosci. 23:10756\u0026ndash;64.\u003c/p\u003e\r\n\r\n\u003cp\u003eSherer TB, Richardson JR, Testa CM, Seo BB, Panov AV, Yagi T, Matsuno-Yagi A, Miller GW, Greenamyre JT (2007) Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson\u0026#39;s disease. J Neurochem. 100(6):1469-79.\u003c/p\u003e\r\n\r\n\u003cp\u003eShulman JM, DeJager PL, Feany MB. 2011. Parkinson\u0026rsquo;s disease: Genetics and Pathogenesis. Annu.Rev.Pathol.Mech.Dis. 6:193-2\u003c/p\u003e\r\n\r\n\u003cp\u003eShults CW. 2004. Mitochondrial dysfunction and possible treatments in Parkinson\u0026rsquo;s disease\u0026ndash;a review. Mitochondrion 4:641\u0026ndash; 648.\u003c/p\u003e\r\n\r\n\u003cp\u003eSinger TP, Ramsay RR.The reaction sites of rotenone and ubiquinone with mitochondrial NADH dehydrogenase. Biochim Biophys Acta. 1994 Aug 30;1187(2):198-202.\u003c/p\u003e\r\n\r\n\u003cp\u003eSpencer DD, Robbins RJ, Naftolin F, Marek KL, Vollmer T, Leranth C, Roth RH, Price LH, Gjedde A, Bunney BS. 1992. Unilateral transplantation of human fetal mesencephalic tissue into the caudate nucleus of patients with Parkinson\u0026#39;s disease. N Engl J Med. 1992 Nov 26;327(22):1541-8\u003c/p\u003e\r\n\r\n\u003cp\u003eSriram K, Matheson JM, Benkovic SA, Miller DB, Luster MI, O\u0026#39;Callaghan JP. 2002. Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: implications for Parkinson\u0026#39;s disease. Faseb J 16(11): 1474-1476.\u003c/p\u003e\r\n\r\n\u003cp\u003eSulzer D, Surmeier DJ. 2013. Neuronal vulnerability, pathogenesis, and Parkinson\u0026rsquo;s disease. Movement Disorders. 28 (6) 715-24.\u003c/p\u003e\r\n\r\n\u003cp\u003eSurmeier DJ1, Guzman JN, Sanchez-Padilla J, Goldberg JA. 2010. What causes the death of dopaminergic neurons in Parkinson\u0026#39;s disease? Prog Brain Res. 2010;183:59-77. doi: 10.1016/S0079-6123(10)83004\u003c/p\u003e\r\n\r\n\u003cp\u003eSilva MA, Mattern C, H\u0026auml;cker R, Tomaz C, Huston JP, Schwarting RK. 1997. Increased neostriatal dopamine activity after intraperitoneal or intranasal administration of L-DOPA: on the role of benserazide pretreatment. Synapse. 27(4):294-302.\u003c/p\u003e\r\n\r\n\u003cp\u003eSzibor M, Gainutdinov T, Fernandez-Vizarra E, Dufour E, Gizatullina Z, Debska-Vielhaber G, Heidler J, Wittig I, Viscomi C, Gellerich F, Moore AL. Bioenergetic consequences from xenotopic expression of a tunicate AOX in mouse mitochondria: Switch from RET and ROS to FET. Biochim Biophys Acta Bioenerg. 2020 Feb 1;1861(2):148137. doi: 10.1016/j.bbabio.2019.148137. Epub 2019 Dec 9. PMID: 31825809.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eSwerdlow RH, Parks JK, Miller SW, Tuttle JB, Trimmer PA, Sheehan JP, Bennett JP Jr, Davis RE, Parker WD Jr (1996) Origin and functional consequences of the complex I defect in Parkinson\u0026#39;s disease. Ann Neurol. 40(4):663-71.\u003c/p\u003e\r\n\r\n\u003cp\u003eTaetzsch T, Block ML. 2013. Pesticides, microglial NOX2, and Parkinson\u0026#39;s disease. J Biochem Mol Toxicol 27(2): 137-149.\u003c/p\u003e\r\n\r\n\u003cp\u003eTansey MG, Goldberg MS. 2009. Neuroinflammation in Parkinson\u0026#39;s disease: Its role in neuronal death and implications for therapeutic intervention. Neurobiol Dis.\u003c/p\u003e\r\n\r\n\u003cp\u003eTebby C, Gao W, Delp J, Carta G, van der Stel W, Leist M, Jennings P, van de Water B, Bois\u0026nbsp;FY. A quantitative AOP of mitochondrial toxicity based on data from three cell lines. Toxicol\u0026nbsp;In Vitro. 2022 Jun;81:105345. doi: 10.1016/j.tiv.2022.105345. Epub 2022 Mar 10. PMID:\u0026nbsp;35278637.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eThomas B., Banerjee R.,Starkova NN., Zhang S., Calingasan NY, Yang L., Wille E., Lorenzo B., Ho D., Beal M., Starkov A. 2012. Mitochondrial permeability transition pore component cyclophilin D distinguishes nigrostriatal dopaminergic death paradigms in the MPTP mouse model of Parkinson\u0026#39;s disease. Antioxidants \u0026amp; redox signaling 16 (9) 855-68\u003c/p\u003e\r\n\r\n\u003cp\u003eThundyil J, Lim KL. 2014. DAMPs and Neurodegeneration. Ageing research reviews.\u003c/p\u003e\r\n\r\n\u003cp\u003eTieu Kim, Imm Jennifer. 2014. Mitochondrial dynamics as potential therapeutic target for Parkinson\u0026rsquo;s disease? ACNR 14 (1) 6-8.\u003c/p\u003e\r\n\r\n\u003cp\u003eTseng YT, Chang FR, Lo YC2. 2014. The Chinese herbal formula Liuwei dihuang protects dopaminergic neurons against Parkinson\u0026#39;s toxin through enhancing antioxidative defense and preventing apoptotic death. Phytomedicine. 21(5):724-33.\u003c/p\u003e\r\n\r\n\u003cp\u003eUitti RJ, Ahlskog JE. 1996. Comparative Review of Dopamine Receptor Agonists in Parkinson\u0026#39;s Disease. C NS Drugs. 5(5):369-88.\u003c/p\u003e\r\n\r\n\u003cp\u003eUitti RJ, Wharen RE Jr, Turk MF, Lucas JA, Finton MJ, Graff-Radford NR, Boylan KB, Goerss SJ, Kall BA, Adler CH, Caviness JN, Atkinson EJ. 1997. Unilateral pallidotomy for Parkinson\u0026#39;s disease: comparison of outcome in younger versus elderly patients. Neurology. 49(4):1072-7.\u003c/p\u003e\r\n\r\n\u003cp\u003evan der Stel W, Carta G, Eakins J, Darici S, Delp J, Forsby A, Bennekou SH, Gardner I, Leist M, Danen EHJ, Walker P, van de Water B, Jennings P. Correction to: Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals. Arch Toxicol. 2020 Aug;94(8):2731-2732. doi: 10.1007/s00204-020-02849-5. Erratum for: Arch Toxicol. 2020 Aug;94(8):2707-2729. doi: 10.1007/s00204-020-02792-5. PMID: 32720191; PMCID: PMC7645484. \u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003evan der Stel, Wanda; Bennekou, Susanne Hougaard; Carta, Giada; Eakins, Julie; Delp, Johannes;\u0026nbsp;Forsby, Anna; Kamp, Hennicke; Gardner, Ian; Zdradil, Barbara; Pastor, Manual (2020) ENV/JM/MONO(2020)22 CASE STUDY ON THE USE OF INTEGRATED APPROACHES TO\u0026nbsp;TESTING AND ASSESSMENT FOR IDENTIFICATION AND CHARACTERISATION OF\u0026nbsp;PARKINSONIAN HAZARD LIABILITY OF DEGUELIN BY AN AOP-BASED TESTING AND READ\u0026nbsp;ACROSS APPROACH Series on Testing and Assessment No. 326\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003evan der Stel W, Yang H, Vrijenhoek NG, Schimming JP, Callegaro G, Carta G, Darici S, Delp J, Forsby A, White A, le D\u0026eacute;v\u0026eacute;dec S, Leist M, Jennings P, Beltman JB, van de Water B, Danen EHJ. Mapping the cellular response to electron transport chain inhibitors reveals selective signaling networks triggered by mitochondrial perturbation. Arch Toxicol. 2022 Jan;96(1):259-285. doi: 10.1007/s00204-021-03160-7. Epub 2021 Oct 13. PMID: 34642769; PMCID: PMC8748354. \u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eVivekanantham S, Shah S, Dewji R,Dewji A, Khatri C \u0026amp; Ologunde R.2014. Neuroinflammation in Parkinson\u0026#39;s disease: role in neurodegeneration and tissue repair. International Journal Of NeuroscienceAccepted. Author Version Posted Online.\u003c/p\u003e\r\n\r\n\u003cp\u003eWalter BL, Vitek JL. 2004. Surgical treatment for Parkinson\u0026#39;s disease. Lancet Neurol. 3(12):719-28.\u003c/p\u003e\r\n\r\n\u003cp\u003eWidner H, Tetrud J, Rehncrona S, Snow B, Brundin P, Gustavii B, Bj\u0026ouml;rklund A, Lindvall O, Langston JW. 1992. Bilateral fetal mesencephalic grafting in two patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). N Engl J Med. 26;327(22):1556-63.\u003c/p\u003e\r\n\r\n\u003cp\u003ewalker DG, Lue LF, Serrano G, Adler CH, Caviness JN, Sue LI, Beach T.2016. altered expression patterns of inflammation-associated and trophic molecules in substantia nigra and striatum brain samples from Parkinson\u0026#39;s disease, incidental Lewy Body disease and normal control cases. Frontiers in Neuroscience. 9:507.\u003c/p\u003e\r\n\r\n\u003cp\u003eWang Q, Chu CH, Oyarzabal E, Jiang L, Chen SH, Wilson B, et al. 2014. Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases. Glia 62(12): 2034-2043.\u003c/p\u003e\r\n\r\n\u003cp\u003eWang T, Zhang W, Pei Z, Block M, Wilson B, Reece JM, et al. 2006. Reactive microgliosis participates in MPP+-induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor. Faseb J 20(7): 906-915.\u003c/p\u003e\r\n\r\n\u003cp\u003eWen Y, Li W, Poteet EC, Xie L, Tan C, Yan LJ, Ju X, Liu R, Qian H, Marvin MA, Goldberg MS, She H, Mao Z, Simpkins JW, Yang SH (2011) Alternative mitochondrial electron transfer as a novel strategy for neuroprotection. J Biol Chem. 286(18):16504-15.\u003c/p\u003e\r\n\r\n\u003cp\u003eWu XF, Block ML, Zhang W, Qin L, Wilson B, Zhang WQ, et al. 2005. The role of microglia in paraquat-induced dopaminergic neurotoxicity. Antioxidants \u0026amp; redox signaling 7(5-6): 654-661.\u003c/p\u003e\r\n\r\n\u003cp\u003eWu RM, Mohanakumar KP, Murphy DL, Chiueh CC. 1994. Antioxidant mechanism and protection of nigral neurons against MPP+ toxicity by deprenyl (selegiline). Ann N Y Acad Sci. 17;738:214-21.\u003c/p\u003e\r\n\r\n\u003cp\u003eYadav S, Gupta SP, Srivastava G, Srivastava PK, Singh MP. 2012. Role of secondary mediators in caffeine-mediated neuroprotection in maneb- and paraquat-induced Parkinson\u0026#39;s disease phenotype in the mouse. Neurochem Res 37(4): 875-884.\u003c/p\u003e\r\n\r\n\u003cp\u003eZaltieri M, Longhena F, Pizzi M, Missale C, Spano P, Bellucci A. 2015. Mitochondrial Dysfunction and \u0026alpha;-Synuclein Synaptic Pathology in Parkinson\u0026#39;s Disease: Who\u0026#39;s on First? Parkinsons Dis. 2015:108029.\u003c/p\u003e\r\n\r\n\u003cp\u003eZhou F, Wu JY, Sun XL, Yao HH, Ding JH, Hu G. 2007. Iptakalim alleviates rotenone-induced degeneration of dopaminergic neurons through inhibiting microglia-mediated neuroinflammation. Neuropsychopharmacology 32(12): 2570-2580.\u003c/p\u003e\r\n\r\n\u003cp\u003eZhu JH, Horbinski C, Guo F, Watkins S, Uchiyama Y, Chu CT. 2007.Regulation of autophagy by extracellular signal-regulated protein kinases during 1-methyl-4-phenylpyridinium-induced cell death. Am. J. Pathol. 170:75\u0026ndash;86.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e- \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003eRevision of AOP3 (Project:\u003c/strong\u003e \u003ca href=\"https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms\" style=\"-webkit-user-drag:none; -webkit-tap-highlight-color:transparent; user-select:text; font-variant-ligatures:normal; white-space:pre-wrap; color:inherit; cursor:text\" target=\"_blank\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#467886\"\u003eNP/EFSA/PREV/2024/02\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/a\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e: \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cem\u003eSearch strings \u003c/em\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eEach strategy followed this structure: Stressor AND Parkinson \u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cem\u003eParkinson\u0026rsquo;s Disease \u0026nbsp;\u003c/em\u003e\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\" dir=\"ltr\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePubMed \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(\u0026quot;Parkinsonian Disorders\u0026quot;[MeSH Terms:noexp] OR \u0026quot;Parkinson Disease\u0026quot;[MeSH Terms] OR \u0026quot;Lewy Body Disease\u0026quot;[MeSH Terms] OR \u0026quot;lewy bodies/pathology\u0026quot;[MeSH Terms] OR \u0026quot;Synucleinopathies\u0026quot;[MeSH Terms:noexp]  OR \u0026quot;Tremor\u0026quot;[MeSH Terms]) OR \u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e(Parkinson* [Title/Abstract] OR paralys*-agitans[Title/Abstract] OR Shaking-pals*[Title/Abstract] OR Synuclein*-Patholog*[Title/Abstract] OR synucleinopathol*[Title/Abstract] OR synuclein*-linked-disease*[Title/Abstract] OR synuclein*-linked-neurodegenerat*[Title/Abstract] OR  synuclein*-linked-neuro-degenerat*[Title/Abstract] OR synuclein-linked*-oligodendrogliopath*[Title/Abstract] OR TREMOR*[Title/Abstract] OR QUIVER*[TITLE/ABSTRACT] OR  \u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e((LEWY*[tiab]) AND (DEMENT*[Title/Abstract] OR DISEASE*[Title/Abstract] OR PATHOL*[TIAB] OR DISORDER*[tiab]))) \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eEMBASE \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(\u0026#39;parkinsonism\u0026#39;/exp OR \u0026#39;Parkinson disease\u0026#39;/de OR \u0026#39;synucleinopathy\u0026#39;/de OR \u0026#39;tremor\u0026#39;/exp OR \u0026#39;diffuse Lewy body disease\u0026#39;/exp  OR \u0026#39;Lewy body\u0026#39;/exp) OR \u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e(Parkinson*:ti,ab,kw OR paralys*-agitans:ti,ab,kw OR Shaking-pals*:ti,ab,kw OR Synuclein*-Patholog*:ti,ab,kw OR synucleinopathol*:ti,ab,kw OR synuclein*-linked-disease*:ti,ab,kw OR synuclein*-linked-neurodegenerat*:ti,ab,kw OR synuclein*-linked-neuro-degenerat*:ti,ab,kw OR synuclein-linked*-oligodendrogliopath*:ti,ab,kw OR TREMOR*:ti,ab,kw OR QUIVER*:ti,ab,kw OR \u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e((LEWY*:ti,ab,kw) AND (DEMENT*:ti,ab,kw OR DISEASE*:ti,ab,kw OR PATHOL*:ti,ab,kw OR DISORDER*:ti,ab,kw))) \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eScopus \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e( ( ( TITLE-ABS-KEY ( lewy* AND ( dement* OR disease* OR pathol* OR disorder* ) ) ) OR ( TITLE-ABS-KEY ( parkinson* OR paralys*-agitans OR shaking-pals* OR synuclein*-patholog* OR synucleinopathol* OR synuclein*-linked-disease* OR synuclein*-linked-neurodegenerat* OR synuclein*-linked-neuro-degenerat* OR synuclein-linked*-oligodendrogliopath* OR tremor* OR quiver* ) ) ) ) \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWeb of Science \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTS=((Parkinson* OR paralys*-agitans OR Shaking-pals* OR Synuclein*-Patholog* OR synucleinopathol* OR synuclein*-linked-disease* OR synuclein*-linked-neurodegenerat* OR synuclein*-linked-neuro-degenerat* OR synuclein-linked*-oligodendrogliopath* OR TREMOR* OR QUIVER*) OR (LEWY* AND (DEMENT* OR DISEASE* OR PATHOL* OR DISORDER*)) and Preprint Citation Index (Exclude \u0026ndash; Database)) \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u003cem\u003eMitochondrial dysfunction\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\" dir=\"ltr\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePubMed \u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e((((((oxygen*[Title/Abstract] OR ROS[Title/Abstract] OR oxidative-stress*[Title/Abstract] OR free-radical*[Title/Abstract] OR superoxide*[Title/Abstract] OR hydrogen-peroxide[Title/Abstract] OR peroxyl-radical*[Title/Abstract] OR electron-transport*[Title/Abstract] OR electron-transfer[Title/Abstract] OR ETC[Title/Abstract] OR respirat*[Title/Abstract] OR oxidative-phosphoryl*[Title/Abstract] OR redox[Title/Abstract] OR bioenerget*[Title/Abstract] OR metabol*[Title/Abstract] OR ATP-synth*[Title/Abstract] OR adenosine-triphosphate-synth*[Title/Abstract] OR catabolism*[Title/Abstract] OR anabolism*[Title/Abstract] OR energ*-product*[Title/Abstract] OR Membran*[Title/Abstract] OR transmembran*[Title/Abstract] OR potential*[Title/Abstract] OR permeabil*[Title/Abstract] OR hypox*[Title/Abstract] OR toxic*[Title/Abstract] OR intox*[Title/Abstract] OR poison*[Title/Abstract] OR contaminat*[Title/Abstract] OR hazard*[Title/Abstract] OR adverse-effect*[Title/Abstract] OR deleteri*[Title/Abstract] OR noxious[Title/Abstract] OR harmful[Title/Abstract] OR detrimental[Title/Abstract] OR oxidation[Title/Abstract]) AND (mitochondr*[Title/Abstract] OR mtDNA[Title/Abstract])) OR (((Mitochondr*[Title/Abstract] OR mtDNA[Title/Abstract] OR Megaconial[Title/Abstract] OR Pleoconial[Title/Abstract] OR Carbamyl-Phosphate-Synth*[Title/Abstract] OR Carbamoylphosphate-Synth*[Title/Abstract] OR CarbamylPhosphate-Synth*[Title/Abstract] OR Carbamoyl-phosphate-Synth*[Title/Abstract] OR CPS-I[Title/Abstract] OR CPS-1[Title/Abstract] OR Cytochrome-c-Oxidas*[Title/Abstract] OR Cox[Title/Abstract] OR Cytochrome-Oxidas*[Title/Abstract] OR Complex[Title/Abstract] OR Multiple-Acyl-CoA-Dehydrogenas*[Title/Abstract] OR Electron-Transfer-Flavoprotein*[Title/Abstract] OR ETFA[Title/Abstract] OR ETFB[Title/Abstract] OR ETFDH[Title/Abstract] OR Pyruvate-Carboxylas*[Title/Abstract] OR PDH[Title/Abstract] OR Pyruvate-Dehydrogenas*[Title/Abstract] OR PDHC[Title/Abstract] OR NADH[Title/Abstract] OR succinate-coenzyme-Q[Title/Abstract] OR succinate-CoQ[Title/Abstract] OR succinate-dehydrogen*[Title/Abstract] OR ubiquinol-cytochrom*[Title/Abstract] OR ubiquin*[Title/Abstract] OR respiratory-chain[Title/Abstract] OR Oxidative-Phosphorylat*[Title/Abstract])) AND (Deficien*[Title/Abstract] OR lack*[Title/Abstract] OR decreas*[Title/Abstract] OR reduct*[Title/Abstract] OR diminut*[Title/Abstract] OR deteriorat*[Title/Abstract] OR Diseas*[Title/Abstract] OR disorder*[Title/Abstract] OR dysfunction*[Title/Abstract] OR Defect*[Title/Abstract] OR insufficien*[Title/Abstract] OR inadequac*[Title/Abstract] OR impair*[Title/Abstract] OR loss[Title/Abstract] OR shortag*[Title/Abstract] OR diminish*[Title/Abstract] OR deplet*[Title/Abstract] OR degenerat*[Title/Abstract] OR Myopath*[Title/Abstract] OR Encephalomyopath*[Title/Abstract] OR syndrom*[Title/Abstract] OR condition*[Title/Abstract] OR abnormal*[Title/Abstract] OR patholog*[Title/Abstract] OR cytopath*[Title/Abstract] OR malfunction*[Title/Abstract] OR anomal*[Title/Abstract] OR damag*[Title/Abstract] OR shrink*[Title/Abstract] OR atroph*[Title/Abstract] OR injur*[Title/Abstract] OR compromis*[Title/Abstract] OR disturb*[Title/Abstract] OR fail*[Title/Abstract] OR breakdown[Title/Abstract] OR declin*[Title/Abstract] OR weaken*[Title/Abstract] OR fragilit*[Title/Abstract] OR instabil*[Title/Abstract]))) OR (Subacute-Necrotizing-Encephalomyel*[Title/Abstract] OR Subacute-Necrotizing-Encephalopath*[Title/Abstract] OR CPEO[Title/Abstract] OR Glutaric-Acidur*[Title/Abstract] OR MADD[Title/Abstract] OR MADDs[Title/Abstract] OR methylmalonic-acidur*[Title/Abstract] OR Ethylmalonic-Adipic-Acidur*[Title/Abstract] OR Ethylmalonic-Adipicacidur*[Title/Abstract] OR Glutaric-Acidem*[Title/Abstract] OR Abnormal-Pyruvate-Metabolism[Title/Abstract] OR Lactic-Acidosis[Title/Abstract] OR mtODE[Title/Abstract] OR oxidative-damage-specific-endonucl*[Title/Abstract] OR MTP[Title/Abstract] OR MOMP[Title/Abstract] OR mitochondriopath*[Title/Abstract] OR MNGIE[Title/Abstract])) OR (Alper*-disease*[Title/Abstract] OR Alper*-syndrom*[Title/Abstract] OR Alpers-Huttenlocher-diseas*[Title/Abstract] OR Alpers-Huttenlocher syndrom*[Title/Abstract])) OR (progressive[Title/Abstract] AND poliodystrophy*[Title/Abstract])) OR ((((\u0026quot;mitochondrial oxidative damage endonuclease\u0026quot; [Supplementary Concept]) OR \u0026quot;Oxygen Consumption\u0026quot;[Mesh]) OR \u0026quot;Mitochondrial Diseases\u0026quot;[Mesh]) OR \u0026quot;Membrane Potential, Mitochondrial\u0026quot;[Mesh]) (Oxygen-Consumpt*[Title/Abstract] OR Anaerobic-Threshold*[Title/Abstract] OR Metabolic-Equivalent*[Title/Abstract])\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u003cem\u003eStressors\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\" dir=\"ltr\" style=\"font-family:-apple-system,BlinkMacSystemFont,\u0026quot;Segoe UI\u0026quot;,Roboto,\u0026quot;Helvetica Neue\u0026quot;,Arial,\u0026quot;Noto Sans\u0026quot;,sans-serif,\u0026quot;Apple Color Emoji\u0026quot;,\u0026quot;Segoe UI Emoji\u0026quot;,\u0026quot;Segoe UI Symbol\u0026quot;,\u0026quot;Noto Color Emoji\u0026quot;; font-size:1rem\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd colspan=\"2\"\u003e\r\n\t\t\t\u003cp\u003eDeguelin\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePubMed \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(\u0026quot;deguelin\u0026quot; [Supplementary Concept] or deguelin*[tiab])\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eEMBASE \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(\u0026#39;deguelin\u0026#39;/exp OR deguelin*:ti,ab,kw,tn)\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eScopus \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e( TITLE-ABS-KEY ( deguelin ) )\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWeb of Science \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(TS=(deguelin)\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd colspan=\"2\"\u003e\r\n\t\t\t\u003cp\u003eFenpyroximate \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePubMed \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(\u0026quot;fenpyroximate\u0026quot; [Supplementary Concept] or fenpyroximat*[tiab])\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eEMBASE \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(\u0026#39;fenpyroximate\u0026#39;/exp OR fenpyroximat*:ti,ab,kw,tn)\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eScopus \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e( TITLE-ABS-KEY ( fenpyroximat* ) )\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWeb of Science \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTS=(fenpyroximat*) \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd colspan=\"2\"\u003e\r\n\t\t\t\u003cp\u003ePyrimidifen \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePubMed \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026quot;pyrimidifen\u0026quot; [Supplementary Concept] OR Pyrimidifen[tiab]\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eEMBASE \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePyrimidifen:ti,ab,kw,tn\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eScopus \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTITLE-ABS-KEY ( piyrimidifen* ) )\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWeb of Science \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTS=(pyrimidifen*)\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd colspan=\"2\"\u003e\r\n\t\t\t\u003cp\u003eTebufenpyrad\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePubMed \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(\u0026quot;4-chloro-N-((4-(1,1-dimethylethyl)phenyl)methyl)-3-ethyl-1-methyl-1H-pyrazole-5-carboxamide\u0026quot; [Supplementary Concept] OR Tebufenpyrad[TIAB] OR\u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003eMK239[TIAB])\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eEMBASE \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(\u0026#39;tebufenpyrad\u0026#39;/exp OR (tebufenpyrad:ti,ab,kw,tn OR mk239:ti,ab,kw,tn))\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eScopus \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e( TITLE-ABS-KEY ( Tebufenpyrad* OR mk239 ) )\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWeb of Science \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e(TS=(Tebufenpyrad* OR mk239)\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n","overall_assessment":"","background":"\u003cp\u003e\u003cstrong\u003eRevision of AOP3 \u003c/strong\u003e(Project: \u003ca href=\"https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms\" rel=\"noreferrer noopener\" target=\"_blank\"\u003eNP/EFSA/PREV/2024/02\u003c/a\u003e):\u003c/p\u003e\r\n\r\n\u003cp\u003eContext \u0026ndash; Revision of AOP3: Inhibition of cI leading to parkinsonian motor deficits\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eIn 2017, the European Food Safety Authority (EFSA) started a project aimed at establishing a mechanistic AOP to elucidate the causal relationship between mitochondrial complex I (cI) inhibition and neurotoxic adverse outcomes manifesting in loss of dopamine neurons (EFSA 2017). This resulted in the development of AOP:3 (inhibition of cI leading to parkinsonian motor deficits), which subsequently was endorsed by the Organisation for Economic Co-operation and Development (OECD). The initial conceptualization of AOP3 was supported by empirical evidence derived from studies involving rotenone and MPTP/MPP+. Subsequent studies explored whether additional mitochondrial cI inhibitors could trigger similar neurotoxic effects (Table 1). In particular, AOP:3 became a test/pilot case to provide an in vitro point-of-departure (PoD) for an AOP-informed Integrated Approach to Testing and Assessment, concerning a potential risk of Parkinsonian motor deficits after exposure to Tebufenpyrad (Alimohammadi 2022). These open literature publications provide a valid source of data for implementing the biological plausibility, empirical support and quantitative characterisation of the AOP 3. Its implementation is being conducted under a negotiated procedure with EFSA (Reference: NP/EFSA/PREV/2024/02), which is intended to update AOP 3 by incorporating additional evidence into the AOP wiki.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eTable 1. Application of AOP 3 in studies with regulatory relevance \u003c/p\u003e\r\n\r\n\u003ctable border=\"1\" dir=\"ltr\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eStudy \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eRegulatory relevance \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eETC tested \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eReference \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTesting of the in vitro battery aligned with AOP3 \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTesting the applicability of several assays to form the basis of a consensus mitochondrial toxicity testing platform \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eInhibition of cI, cII or cIII \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eDelp et al. 2019; van der Stel et al. 2020 \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eCase study on the use of an IATA for identification and characterization of Parkinsonian hazard\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eRead across safety assessment of structurally closely related mitochondrial cI inhibitors\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eInhibition of cI\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eENV/JM/MONO(2020)22,\u0026nbsp;\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003evan der Stel, W. (2021)\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd colspan=\"1\" rowspan=\"2\"\u003e\r\n\t\t\t\u003cp\u003eTesting the predictivity of the downstream events \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTesting the inhibitory potency prediction with the aim to understand how far early KE data can and will predict an AO \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eInhibition of cI, cII or cIII \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eDelp et al., 2021 \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eCalibrate a qAOP to predict downstream KEs\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ecI inhibitors\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTebby et al., 2022\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eEFSA Pilot Project on New Approach Methodologies\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMargins of Internal Exposure Application to Estimated Brain Exposure Compared to In Vitro PoD\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ecI inhibitor\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eAlimohammadi et al. 2023\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eHazard assessment \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eIdentification of the signalling network triggered by mitochondrial perturbation induced by the inhibition of cI, cII or cIII in HepG2 cells \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eInhibition of cI, cII or cIII \u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eVan der Stel et al., 2022 \u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003ePoD: Poin t of Departure; IATA: Integrated Approaches to Testing and Assessment; cI: complex, I; cII: complex II; cIII: complex III\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003eNot endorsed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n","user_defined_mie":"888: Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I)","user_defined_ao":"896: Parkinsonian motor deficits","oecd_project":"1.33","oecd_status_id":1,"graphical_representation_image_uid":"2018/02/25/1vwk4p05mb_4rkk0gx9a1_AOP3NewSchema_correct.png","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2025-10-04T02:11:56.000-04:00","development_strategy":"\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e- Revision of AOP3 (Project:\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e \u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003ca href=\"https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms\" style=\"color:#467886; text-decoration:underline\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003eNP/EFSA/PREV/2024/02\u003c/span\u003e\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e):\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;The starting conceptual model for this project is based on the key scientific sources reported in table 1. These publications provided the initial evidence for this project, which was further expanded through a structured literature review aimed at updating the link between mitochondrial toxicity and parkinsonian motor deficits across AOP 3. The updates have been documented in a dedicated section, in agreement with the AOP3 point of contact.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eFor well-established MIEs and KEs, evidence was retrieved from seminal publications recommended by domain experts and supplemented by expert knowledge.  Additional literature was identified, through a structured, non-systematic search using a stressor-based search strategy to retrieve essentiality data and data linking KE887/KE177 to 890/AO through the selected stressors in in vivo studies. Tailored search strings, detailed in a dedicated section at the end of this document, were designed by two information specialists in collaboration with the project team. For each selected stressor, the information specialists conducted a literature search using a quasi-systematic approach. They employed both textwords and database-specific subject headings where available, across the following databases: PubMed, Embase via Elsevier, Web of Science via Clarivate, and Scopus.  \u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eThe following criteria were applied to select relevant studies. \u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003ePublication type\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003ctable cellspacing=\"0\" class=\"Table\" style=\"border-collapse:collapse; width:0px\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#275317; border-bottom:4px solid white; border-left:1px solid white; border-right:1px solid white; border-top:1px solid white; height:20px; vertical-align:top; width:105px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eTime\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:4px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:20px; vertical-align:top; width:42px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:4px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:20px; vertical-align:top; width:484px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eInception \u0026ndash; present or\u0026nbsp; 2017- present\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#275317; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:105px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eLanguage\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:42px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:484px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eEnglish\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#275317; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:105px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003ePublication type\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:42px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:484px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Primary research studies\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Reviews\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#275317; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:105px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:42px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eOUT\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:484px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Expert opinions\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;editorials\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;letters to the editor\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;conference proceedings and posters\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;retracted articles\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;PhD thesis\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003eIn vitro studies\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003ctable cellspacing=\"0\" class=\"Table\" style=\"border-collapse:collapse; width:0px\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#275317; border-bottom:4px solid white; border-left:1px solid white; border-right:1px solid white; border-top:1px solid white; height:20px; vertical-align:top; width:103px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eStudy design\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:4px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:20px; vertical-align:top; width:41px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:4px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:20px; vertical-align:top; width:487px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eAny In vitro study design\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd rowspan=\"2\" style=\"background-color:#275317; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:103px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003ePopulation\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:41px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:487px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Only cells of the nervous system (i.e., neuronal population and glial cells) at a mature stage\u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;All species\u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:41px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eOUT\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:487px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eAll except those included\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd rowspan=\"2\" style=\"background-color:#275317; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:103px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eExposure\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:41px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:487px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Identified stressors (Objective 2)\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;The exposure must occur during the mature stage\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:41px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eOUT\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:487px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Chemical mixture\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Less than one control and three concentrations tested\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#275317; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:103px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eEndpoints\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:41px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:487px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;ETC inhibition\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Mitochondrial dysfunction (i.e., oxygen consumption rate, mitochondrial membrane potential, elevated reactive oxygen species, mitochondrial oxidative damage)\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Degeneration of dopaminergic neurons\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003eIn vivo studies\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003ctable cellspacing=\"0\" class=\"Table\" style=\"border-collapse:collapse; width:0px\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd rowspan=\"2\" style=\"background-color:#275317; border-bottom:4px solid white; border-left:1px solid white; border-right:1px solid white; border-top:1px solid white; height:20px; vertical-align:top; width:111px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eStudy design\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:4px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:20px; vertical-align:top; width:36px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:4px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:20px; vertical-align:top; width:482px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eAny\u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:36px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eOUT\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:482px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eNone\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd rowspan=\"2\" style=\"background-color:#275317; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:111px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003ePopulation\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:36px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:482px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMammals and zebrafish\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:36px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eOUT\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:482px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eAll except those included\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd rowspan=\"2\" style=\"background-color:#275317; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:111px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eExposure\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:36px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:482px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Identified stressors.\u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;The exposure must occur in adults.\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:36px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eOUT\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#ccd2d8; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:482px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;In-uterus, developmental stage.\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Mixtures.\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Less than one control and two concentrations tested\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#275317; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:111px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eEndpoints\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:36px\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIN\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#e7eaed; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:20px; vertical-align:top; width:482px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Labeling of dopaminergic neurons by fluorescent dopamine analogs, or genetically labeled dopaminergic neurons (e.g., GFP expression under control of TH promoter).\u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Degeneration of dopaminergic neurons\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:36px; text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026middot;Motor deficits\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Times New Roman\u0026quot;,serif\"\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003eTo develop the empirical evidence, chemicals listed in Delp 2019 and Delp 2021 were considered. In addition, for compounds that were identified or measured as cI inhibitor, data were extracted from \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003eAlimohammadi et al. (2023); van der Stel-OECD\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003e (2020)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003e, van der Stel, W. (2021)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003e,\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003e Van der Stel et al., (2022)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003e and Tebby et al. (2022)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003e. \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#444444\"\u003eEndpoints and assays were selected on their relevance to AOP 3 and the use of appropriate cell models (i.e., neuronal cells and HepG2 for KE1, neuronal cells only for KE2 and KE4).\u0026nbsp; Further details are provided in the KE section \u0026ldquo;How it is measured\u0026rdquo; and in the empirical evidence for the KER.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-size:11.0pt\"\u003e\u003cspan style=\"background-color:white\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eQuantitative understanding of the KERs was gained by modelling the KERs within the qAOP framework and methods that were developed in Tebby et al. (2022) and further developed during the negotiated procedure with EFSA (Reference: NP/EFSA/PREV/2024/02). A set of compounds used for AOP quantification was selected based on availability of multiple-concentration data representing at least two identical adjacent KEs. Equations representing the KERs were selected based on the dose-response data for adjacent KEs. These equations were parameterized using a bayesian framework, which allowed completing data gaps for cIII inhibition with prior knowledge on cI inhibition.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Tahoma\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:#212529\"\u003e- Not endorsed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n","known_modulating_factors":"\u003cdiv\u003e\r\n\u003ctable class=\"table table-bordered table-fullwidth\"\u003e\r\n\t\u003cthead\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003cth\u003eModulating Factor (MF)\u003c/th\u003e\r\n\t\t\t\u003cth\u003eInfluence or Outcome\u003c/th\u003e\r\n\t\t\t\u003cth\u003eKER(s) involved\u003c/th\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/thead\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\u003c/div\u003e\r\n","assigned_license_id":2,"handbook_id":1,"project_129":false},{"id":4,"title":"Ecdysone receptor agonism leading to mortality via suppression of Ftz-f1","short_name":"EcR agonism leading to mortality via suppression of Ftz-f1","corresponding_author_id":184,"abstract":"\u003cp\u003eThis Adverse Outcome Pathway (AOP) describes how hyperactivation of the ecdysone receptor (EcR) in arthropods can lead to lethal molting disruption and mortality. Binding of natural ligands (ecdysteroids) to EcR is critical for regulating molting and metamorphosis in insects and crustaceans. However, inappropriate or prolonged activation of EcR by exogenous chemicals disrupts the tightly regulated temporal gene expression cascade required for successful ecdysis. Key events (KEs) include altered expression of early transcription factors (E75B, Ftz-f1), reduction of circulating neuropeptides (CCAP, ETH), impaired motor program activity, and suppression of abdominal muscle contraction, ultimately resulting in incomplete ecdysis and death. This AOP provides mechanistic understanding relevant for environmental chemical safety assessment, particularly regarding pesticides and other compounds targeting insect endocrine systems.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:15.000-05:00","updated_at":"2025-10-02T04:18:29.000-04:00","status_id":1,"authors":"\u003cp\u003eYou Song\u0026nbsp;and Knut Erik Tollefsen\u003cbr /\u003e\r\nNorwegian Institute for Water Research (NIVA), \u0026Oslash;kernveien 94, N-0579 Oslo, Norway\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eTaxa:\u003c/strong\u003e Arthropods, primarily insects (Diptera, Lepidoptera, Coleoptera) and crustaceans.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eLife stage:\u003c/strong\u003e Juvenile and larval stages undergoing molting.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eSex:\u003c/strong\u003e Both sexes are equally affected, as molting regulation is not sex-specific.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eOther considerations:\u003c/strong\u003e The pathway is most relevant in holometabolous insects but is applicable across arthropods where molting is controlled by EcR signaling.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","key_event_essentiality":"\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEcR hyperactivation (MIE):\u003c/strong\u003e Genetic or pharmacological overactivation prevents correct timing of molting cascades, leading to lethality.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eE75B and Ftz-f1 expression changes:\u003c/strong\u003e Knockout or overexpression experiments demonstrate disruption of subsequent endocrine signals and molting success.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eCirculating ETH/CCAP:\u003c/strong\u003e Blocking or reducing peptide release suppresses ecdysis behavior. Rescue experiments with exogenous peptides restore motor program activity.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eMotor program activity and abdominal contractions:\u003c/strong\u003e Neurophysiological studies show that impaired muscle activity directly prevents exuviation.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eIncomplete ecdysis:\u003c/strong\u003e Universally essential for linking endocrine dysfunction to mortality.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003eOverall, essentiality of KEs is supported by direct experimental evidence in multiple model arthropods.\u003c/p\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003e\u003cstrong\u003eKER 103 \u0026rarr; 1264 (EcR hyperactivation \u0026rarr; Increased E75B expression)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eBiological plausibility:\u003c/strong\u003e Strong. E75B is a primary-response gene in the ecdysone signaling cascade. Overactivation of EcR drives prolonged or elevated expression of E75B.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEmpirical support:\u003c/strong\u003e Multiple in vitro and in vivo studies (e.g., \u003cem\u003eDrosophila\u003c/em\u003e, lepidopterans) show dose-dependent induction of E75B following EcR agonist exposure.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eUncertainties:\u003c/strong\u003e Quantitative thresholds vary across taxa.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eKER 103 \u0026rarr; 1265 (EcR hyperactivation \u0026rarr; Decreased Ftz-f1 expression)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eBiological plausibility:\u003c/strong\u003e Strong. Ftz-f1 is normally induced after a decline in ecdysone signaling, serving as a competence factor for subsequent developmental transitions. Sustained EcR activity suppresses Ftz-f1 expression.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEmpirical support:\u003c/strong\u003e Genetic experiments in \u003cem\u003eDrosophila\u003c/em\u003e demonstrate that EcR hyperactivation prevents Ftz-f1 induction, leading to molting defects.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eUncertainties:\u003c/strong\u003e The precise timing of downregulation differs between insect species.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eKER 1265 \u0026rarr; 998 (Decreased Ftz-f1 expression \u0026rarr; Decreased circulating ETH)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eBiological plausibility:\u003c/strong\u003e Moderate to strong. ETH release from Inka cells requires proper transcriptional programming, in which Ftz-f1 plays a permissive role.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEmpirical support:\u003c/strong\u003e ETH levels are reduced in Ftz-f1 mutant or RNAi knockdown insects, with corresponding ecdysis failure.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eUncertainties:\u003c/strong\u003e Direct mechanistic links in crustaceans are less studied.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eKER 1264 \u0026rarr; 1266 (Increased E75B expression \u0026rarr; Decreased circulating CCAP)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eBiological plausibility:\u003c/strong\u003e Moderate. E75B dysregulation affects downstream neural peptide release patterns, including crustacean cardioactive peptide (CCAP).\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEmpirical support:\u003c/strong\u003e Studies in \u003cem\u003eManduca sexta\u003c/em\u003e and \u003cem\u003eDrosophila\u003c/em\u003e show disrupted CCAP neuron activation in response to EcR agonists.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eUncertainties:\u003c/strong\u003e Empirical dose-response data linking E75B overexpression directly to CCAP suppression are limited.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eKER 1266 \u0026rarr; 1267 (Decreased circulating CCAP \u0026rarr; Decreased ecdysis motor program activity)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eBiological plausibility:\u003c/strong\u003e Strong. CCAP is required to initiate and maintain the motor patterns driving ecdysis behavior.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEmpirical support:\u003c/strong\u003e Ablation or silencing of CCAP neurons abolishes normal ecdysis behavior in insects. Exogenous CCAP restores motor program activity in some experimental systems.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eUncertainties:\u003c/strong\u003e Quantitative thresholds for peptide levels triggering full motor program are not well established.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eKER 998 \u0026rarr; 993 (Decreased circulating ETH \u0026rarr; Decreased abdominal muscle contraction)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eBiological plausibility:\u003c/strong\u003e Strong. ETH directly triggers ecdysis motor output by activating central nervous system circuits.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEmpirical support:\u003c/strong\u003e ETH knockout or peptide inhibition prevents abdominal contractions in \u003cem\u003eDrosophila\u003c/em\u003e larvae. ETH injection can rescue the phenotype.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eUncertainties:\u003c/strong\u003e Effects may be stage-dependent.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eKER 1267 \u0026rarr; 990 (Decreased ecdysis motor program activity \u0026rarr; Incomplete ecdysis)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eBiological plausibility:\u003c/strong\u003e Strong. Motor program activity is essential for successful shedding of the old cuticle.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEmpirical support:\u003c/strong\u003e Neurophysiological and behavioral studies show that impaired motor activity directly correlates with failed or incomplete ecdysis.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eUncertainties:\u003c/strong\u003e Variation in motor outputs among species may influence severity.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eKER 993 \u0026rarr; 990 (Decreased abdominal muscle contraction \u0026rarr; Incomplete ecdysis)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eBiological plausibility:\u003c/strong\u003e Strong. Abdominal contractions generate the mechanical force required for cuticle shedding.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEmpirical support:\u003c/strong\u003e Pharmacological or genetic inhibition of abdominal muscle contraction prevents complete ecdysis in \u003cem\u003eDrosophila\u003c/em\u003e and other insects.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eUncertainties:\u003c/strong\u003e Contribution of other body muscles (e.g., thoracic) not fully quantified.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eKER 990 \u0026rarr; 350 (Incomplete ecdysis \u0026rarr; Increased mortality)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eBiological plausibility:\u003c/strong\u003e Strong. Inability to shed the old cuticle is incompatible with survival.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eEmpirical support:\u003c/strong\u003e High mortality rates are consistently observed in laboratory and field studies where molting is disrupted by EcR agonists or neuropeptide blockers.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eUncertainties:\u003c/strong\u003e Mortality timing (immediate vs delayed) may vary with species and stage.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","quantitative_considerations":"\u003cp\u003eQuantitative relationships between EcR activation and downstream transcriptional responses are partially characterized, especially in \u003cem\u003eDrosophila\u003c/em\u003e. Dose-response data for diacylhydrazine insecticides provide empirical linkage between exposure and incomplete ecdysis. However, quantitative models are still limited and largely taxon-specific.\u003c/p\u003e\r\n","optional_considerations":"\u003cp\u003eThis AOP has clear utility for both scientific and regulatory applications related to the environmental assessment of endocrine-active substances targeting arthropod molting. Because EcR is the primary molecular target of many insect growth regulators (IGRs), this pathway provides a mechanistic framework for interpreting how chemical binding at the receptor level translates to population-level adverse outcomes such as mortality.\u003c/p\u003e\r\n\r\n\u003cp\u003eFrom a regulatory perspective, the AOP can inform the development and refinement of OECD test guidelines addressing arthropod development and molting. It can also support the design of integrated approaches to testing and assessment (IATA), in which data from in vitro receptor-binding assays, transcriptomic biomarkers (e.g., E75B, Ftz-f1), and neuropeptide measurements can be combined with higher-tier organismal studies to streamline hazard characterization. Furthermore, the pathway may facilitate the identification of molecular biomarkers that can be incorporated into early screening assays, reducing reliance on animal-intensive in vivo tests.\u003c/p\u003e\r\n\r\n\u003cp\u003eThe AOP is also relevant for chemical grouping and read-across approaches, particularly for compounds within the diacylhydrazine class and other EcR agonists. Structure\u0026ndash;activity relationship ((Q)SAR) models or chemical profilers trained on these endpoints could help predict EcR activity and prioritize substances for further testing.\u003c/p\u003e\r\n\r\n\u003cp\u003eFor ecological risk assessment, this AOP highlights the potential for population-level impacts on non-target arthropods, including beneficial insects (e.g., pollinators) and aquatic crustaceans. Given the essential role of molting for growth and reproduction, disruptions captured in this pathway provide a mechanistic basis to link molecular initiating events to ecologically relevant endpoints.\u003c/p\u003e\r\n\r\n\u003cp\u003eOverall, this AOP offers opportunities to improve chemical safety decision-making by providing a structured framework to integrate mechanistic data into regulatory contexts, enabling screening, prioritization, and risk assessment of chemicals that act through EcR hyperactivation.\u003c/p\u003e\r\n","references":"\u003cp style=\"margin-left:12.9pt\"\u003eSong, Y.; Villeneuve, D. L.; Toyota, K.; Iguchi, T.; Tollefsen, K. E., 2017. \u003cstrong\u003eEcdysone receptor agonism leading to lethal molting disruption in arthropods: review and adverse outcome pathway development\u003c/strong\u003e. Environ Sci Technol, 51, (8), 4142-4157.\u003c/p\u003e\r\n\r\n\u003cp style=\"margin-left:12.9pt\"\u003eSong, Y., Evenseth, L.M., Iguchi, T., Tollefsen, K.E., 2017. \u003cstrong\u003eRelease of chitobiase as an indicator of potential molting disruption in juvenile \u003cem\u003eDaphnia magna\u003c/em\u003e exposed to the ecdysone receptor agonist 20-hydroxyecdysone\u003c/strong\u003e. J Toxicol Environ Health A, 1-9\u003c/p\u003e\r\n\r\n\u003cp style=\"margin-left:12.9pt\"\u003eFay, K. A., Villeneuve, D. L., LaLone, C. A., Song, Y., Tollefsen, K. E. and Ankley, G. T., 2017. \u003cstrong\u003ePractical approaches to adverse outcome pathway (AOP) development and weight of evidence evaluation as illustrated by ecotoxicological case studies\u003c/strong\u003e. Environ. Toxicol. Chem. 36(6):1429-1449.\u003c/p\u003e\r\n\r\n\u003cp style=\"margin-left:12.9pt\"\u003eMiyakawa, H., Sato, T., Song, Y., Tollefsen, K.E., Iguchi, T., 2017. \u003cstrong\u003eEcdysteroid and juvenile hormone biosynthesis, receptors and their signaling in the freshwater microcrustacean \u003cem\u003eDaphnia\u003c/em\u003e\u003c/strong\u003e. J Steroid Biochem Mol Biol. pii: S0960-0760(17), 30370-30379.\u003c/p\u003e\r\n\r\n\u003cp style=\"margin-left:12.9pt\"\u003e\u0026nbsp;\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003eThe overall weight of evidence supporting this AOP is strong, with high biological plausibility and multiple lines of empirical support linking EcR hyperactivation to lethal molting disruption. The key endocrine and neuropeptide signaling pathways involved are highly conserved across arthropods, increasing confidence in broad taxonomic applicability, particularly to insects and crustaceans undergoing ecdysis. Essentiality of key events is well demonstrated through genetic and pharmacological manipulations, and direct causal linkages between upstream molecular initiating events and downstream organism-level outcomes are supported by both in vitro and in vivo studies. While quantitative understanding remains incomplete, especially regarding cross-species dose-response relationships, the evidence base is sufficient to support application in chemical screening, prioritization, and risk assessment. The AOP is considered reliable for use in evaluating the hazards of EcR agonists and related endocrine-active chemicals, with clear regulatory relevance to the assessment of insect growth regulators and protection of non-target arthropods.\u003c/p\u003e\r\n","background":"\u003cp\u003eThe development of this AOP is motivated by regulatory needs to understand and predict the impacts of insect growth regulators and other endocrine-active chemicals that target EcR. Such compounds are widely used in pest control but pose risks to non-target arthropods, including pollinators and aquatic invertebrates. The AOP formalizes knowledge of conserved molting endocrine pathways to support hazard identification and potential regulatory screening frameworks.\u003c/p\u003e\r\n","user_defined_mie":"103: Increase, Ecdysone receptor agonism","user_defined_ao":"","oecd_project":"","oecd_status_id":null,"graphical_representation_image_uid":"2025/09/29/9mhujlly8j_Picture1.png","saaop_status_id":3,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2025-10-02T04:18:29.000-04:00","development_strategy":"\u003cp\u003eThe AOP was developed based on structured literature reviews and expert knowledge. Key sources included primary research on EcR signaling, molting neuropeptides (ETH, CCAP), transcriptional cascades in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e and other model insects, as well as crustacean endocrinology. Literature searches were conducted in PubMed, Web of Science, and Scopus using terms such as \u003cem\u003eecdysone receptor agonists\u003c/em\u003e, \u003cem\u003eecdysis motor program\u003c/em\u003e, \u003cem\u003einsect molting disruption\u003c/em\u003e, \u003cem\u003e20-hydroxyecdysone\u003c/em\u003e, and \u003cem\u003eecdysteroid signaling\u003c/em\u003e. Priority was given to studies demonstrating experimental perturbation of EcR or downstream KEs and their effects on molting success and survival. Reviews and AOP frameworks (e.g., OECD guidance) were used to ensure structured evaluation and alignment with regulatory relevance.\u003c/p\u003e\r\n","known_modulating_factors":"\u003cdiv\u003e\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eTemperature:\u003c/strong\u003e Affects hormone turnover and molting periodicity.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eNutritional status:\u003c/strong\u003e Influences steroid hormone synthesis and peptide release.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eSpecies-specific sensitivity:\u003c/strong\u003e Different insects and crustaceans vary in susceptibility to EcR agonists.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli\u003e\r\n\t\u003cp\u003e\u003cstrong\u003eDevelopmental stage:\u003c/strong\u003e Early versus late larval stages may have differing vulnerability.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003ctable class=\"table table-bordered table-fullwidth\"\u003e\r\n\t\u003cthead\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003cth\u003eModulating Factor (MF)\u003c/th\u003e\r\n\t\t\t\u003cth\u003eInfluence or Outcome\u003c/th\u003e\r\n\t\t\t\u003cth\u003eKER(s) involved\u003c/th\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/thead\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\u003c/div\u003e\r\n","assigned_license_id":3,"handbook_id":1,"project_129":false},{"id":6,"title":"Antagonist binding to PPARα leading to body-weight loss","short_name":"PPARα antagonism leading to body-weight loss","corresponding_author_id":329,"abstract":"\u003cp\u003eThe present AOP describes antagonistic chemical binding to the peroxisome proliferator-activated receptor \u0026alpha; (PPAR\u0026alpha;) resulting in preferential binding a co-repressor to the overall PPAR\u0026alpha; signaling complex causing a chain of events that includes: antagonism of PPAR\u0026alpha; nuclear signaling, decreased transcriptional expression of PPAR\u0026alpha;-regulated genes that support energy metabolism, and inhibited metabolic energy production culminating with starvation-like weight loss. The AOP is likely to be synergized during fasting, starvation or malnutrition events.\u0026nbsp; The MIE for this AOP involves antagonistic PPAR\u0026alpha; binding. The antagonist-binding to the PPAR\u0026alpha; regulatory complex causes the KE1, stabilization of co-repressor (SMRT or N-CoR) to PPARalpha ligand binding domain suppressing PPAR\u0026alpha; nuclear signaling (Nagy et al 1999, Xu et al 2002). PPAR\u0026alpha; is a transcriptional regulator for a variety of genes that facilitate systemic energy homeostasis (Mirza et al 2019, Kersten 2014, Evans et al 2004, Desvergne and Wahli 1999). As a result of the MIE and then KE1, the KE2 occurs where PPARalpha transactivation is inhibited for genes involved in the next 2 key events of the AOP: (KE3) decreased fatty acid beta oxidation (Desvergne and Wahili 1999, Kersten 2014, Dreyer et al 1992, Lazarow 1978, Brandt et al 1998; Mascaro et al 1998, Aoyama et al 1998, Gulick et al 1994, Sanderson et al 2008) and (KE4) decreased ketogenesis (Cahil 2006, Kersten et al 2014, Sengupta et al 2010, Desvergne and Wahli 1999). The KE3 results in decreased catabolism of very long chain fatty acids in peroxisomes and reduced catabolism of long, medium and short chain fatty acids in mitochondria reducing acetyl-CoA availability for use in oxidative phosphorylation-based ATP production (Evans et al 2004).\u0026nbsp; KE2 (and also potentially KE3) can drive KE4 resulting in decreased potential to repackage energy substrates as ketone bodies to support systemic energy demands during periods where the systemic energy budget is negative (Badman et al 2007, Potthoff 2009; Muoio et al 2002). The KE5, no change or a decrease in circulating ketone bodies becomes critical during cellular energy deficit conditions, a state where ketogenesis is typically induced to increase circulating ketone bodies providing metabolic fuel to sustain energy homeostasis (Cahill 2006). Physiological studies of the progression of human starvation have demonstrated the critical importance of ketogenesis, especially production of \u0026beta;-hydroxybutyrate, for meeting systemic energy demands by supplementing glucose to sustain the energy requirements of the brain (Cahill 2006, Owen et al 2005). PPAR\u0026alpha; knock can inhibit ketogenesis from fatty acid substrates in fasted mice reducing \u0026beta;-hydroxybutyrate production causing hypoketonemia (Badman et al 2007, Le May et al 2000, Muoio et al 2002).\u0026nbsp; Sustained negative energy budgets lead to KE6, an increase in muscle protein catabolism, with glutamine and alanine recycled for gluconeogenesis (Felig et al 1970A, Kashiwaya et al 1994). \u0026nbsp;If ketogenesis from fatty acid substrates fails to meet cellular energy needs, gluconeogenesis from alternative substrates becomes necessary including (KE 6) muscle protein catabolism \u003cem\u003ein situ\u003c/em\u003e supporting local muscle function and releasing glutamine (Marliss et al 1971) and alanine (Felig et al 1970A) for gluconeogenesis in kidney and liver to sustain systemic energy needs (Goodman et al 1966, Kashiwaya et al 1994, Cahill 2006).\u0026nbsp; Finally, the AO of body-weight loss occurs, which within the context of dynamic energy budget theory, decreases energy allocations to organismal maturation and reproduction (Nisbet et al 2000) and has been demonstrated to negatively affect ecological fitness (Martin et al 1987).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:15.000-05:00","updated_at":"2023-04-29T16:02:54.000-04:00","status_id":1,"authors":"\u003cp\u003e\u003cstrong\u003eKurt A. Gust\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e, Mitchell S. Wilbanks\u003csup\u003e1\u003c/sup\u003e, Zachary A. Collier\u003csup\u003e1\u003c/sup\u003e, Lyle D. Burgoon\u003csup\u003e1\u003c/sup\u003e, Edward J. Perkins\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003e1Army Engineer Research and Development Center, Vicksburg, MS, 39180, Kurt.A.Gust@usace.army.mil; Mitchell.S.Wilbanks@usace.army.mil;\u003c/p\u003e\r\n\r\n\u003cp\u003ePoint of Contact: Kurt A. Gust, Kurt.A.Gust@usace.army.mil\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003eThe majority of the evidence described in this AOP are derived for either human (mostly in vitro) or mice (in vivo and in vitro) studies.\u0026nbsp; There are recognized differences between mouse and human PPAR\u0026alpha; signaling and responses from the literature, however, for our specific KEs, the responses among species are relatively well conserved.\u0026nbsp; Therefore, we have reasonable confidence that the AOP provides reliable confidence for human health assessment.\u0026nbsp; The AOP also has the potential to support ecotoxicological assessment if there is reasonable confidence that the KEs are conserved in the species of interest.\u003c/p\u003e\r\n","key_event_essentiality":"\u003cp\u003eRationale for essentiality calls:\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eMIE:\u0026nbsp; PPAR\u0026alpha;, Binding of antagonist:\u0026nbsp; \u003c/strong\u003eRegarding the present MIE, molecules can bind to the PPAR\u0026alpha; regulatory complex affecting the binding of co-activators and co-repressors. Specifically designed molecules such as the PPAR\u0026alpha; antagonists GW6471 can bind to PPAR\u0026alpha; selectively recruiting binding of co-repressors to the PPAR\u0026alpha; nuclear signaling complex (Xu et al 2002).\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eKey Event 1:\u0026nbsp; PPAR alpha co-repressor, Increased \u003c/strong\u003e- The binding of co-repressors to the PPAR\u0026alpha; signaling complex suppresses nuclear signaling and thus downstream transcription of PPAR\u0026alpha;-regulated genes (Liu et al 2008).\u0026nbsp; GW6471 binding to the co-repressor is reversible thus allowing the co-repressor to leave the ligand binding domain of PPAR\u0026alpha;, restoring normal function (Xu et al 2002).\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eKey Event 2:\u0026nbsp; PPARalpha transactivation of gene expression, Decreased \u003c/strong\u003e- As described in a variety of reviews, PPARalpha represents a master regulator of energy metabolism which specifically promotes fatty oxidation for energy production \u0026amp; distribution (Evans et al 2004, Kersten 2014, Lefebvre et al 2006, Desvergne and Wahili 1999, Mirza et al 2019).\u0026nbsp; Both PPARalpha knock outs and PPARalpha antagonism decreased transcriptional expression of gene targets involved in peroxisomal fatty acid beta oxidation (Kersten et al 1999, Desvergne and Wahili 1999, Janssen et al 2015), mitochondrial fatty acid beta oxidation (Brandt et al 1998; Mascaro et al 1998, Kersten\u0026nbsp; 2014), and ketogenesis (Sengupta et al 2010, Desvergne and Wahli 1999, Kersten 2014).\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eKey Event 3: Fatty Acid Beta Oxidation, Decrease\u003c/strong\u003e\u003cstrong\u003ed \u0026ndash;\u003c/strong\u003e This key event is essential for deriving metabolic energy from fatty acid substrates thus supporting a large component of overall organismal energy demands (Evans et al 2004, Kersten 2014, Desvergne and Wahili 1999).\u0026nbsp; Very long chain fatty acids (\u0026gt;C20) are metabolized in the peroxisome and short, medium and long chain fatty acids (\u0026lt;C20) are catabolized by mitochondrial beta-oxidation.\u0026nbsp; PPARalpha regulates nearly every enzymatic step in the uptake as well as the oxidative breakdown of acyl-CoAs to acetyl-CoA (Kersten 2014).\u0026nbsp; The acetyl-CoA monomers serve as fundamental units for metabolic energy production (ATP) via the citric acid cycle followed by electron-transport chain mediated oxidative phosphorylation (Nelson and Cox, 2000A) as well as serve as the fundamental units for energy storage via gluconeogenesis (Nelson and Cox, 2000B) and lipogenesis (Nelson and Cox, 2000C).\u0026nbsp; PPARalpha knockout studies have demonstrated impaired mitochondrial fatty acid oxidation leading to fatty acid accumulation in the liver (Badmann et al 2007) as well as an inability to meet systemic energy demands (Kersten et al, 1999).\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eKey Event 4: Ketogenesis (production of ketone bodies), decrease\u003c/strong\u003e\u003cstrong\u003ed - \u003c/strong\u003eThe liver represents a key organ involved in systemic energy distribution given its ability to synthesize glucose (an ability shared only with the kidney, Gerich et al 2001) as well as its exclusive role in the generation of ketone bodies (Cahill 2006, Sengupta et al 2010, Kersten 2014).\u0026nbsp; This is especially important for the metabolic energy needs of the brain which can only use glucose and the ketone body, \u0026beta;-hydroxybutyrate for cellular energy production (Cahill 2006, Owen 2005, Kersten 2014).\u0026nbsp; Therefore, ketogenesis is critical to supporting general systemic energy homeostasis in fasting events (Cahill 2006, Evans et al 2004, Sengupta et al 2010).\u0026nbsp; Interference with ketogenesis, for example by PPAR\u0026alpha; inhibition, has been demonstrated to inhibit \u0026beta;-hydroxybutyrate production (measured in serum) during fasting events in mice (Le May et al 2000, Badman et al 2007, Potthoff 2009, Sengupta et al 2010) and cause hypoketonemia (Muoio et al 2002).\u0026nbsp; The Badman et al (2007) study indicated that metabolism of fatty acid substrates (measured as liver triglycerides) that would otherwise contribute to \u0026beta;-hydroxybutyrate production was additionally inhibited under PPAR\u0026alpha; knockout.\u0026nbsp;\u0026nbsp;\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eKey Event 5: Circulating Ketone Bodies, Not Increased - \u003c/strong\u003ePhysiological studies of the progression of human starvation have identified that the preferred metabolic fuel is glucose in the fed state and progressing through two days of fasting, afterward ketone bodies become increasingly important for meeting energy demands (Cahill 2006, Owen et al 2005).\u0026nbsp; Substrates derived from carbohydrates, fats and protein can contribute to gluconeogenesis (Cahill 2006, Gerich et al 2001) whereas substrates derived from fatty acids are the primary contributors to ketogenesis (Desvergne and Wahli 1999).\u0026nbsp; Cahill (2006) and colleagues have demonstrated the importance of ketone body production, especially \u0026beta;-hydroxybutyrate, for maintaining energy homeostasis during starvation by serving as an alternative substrate to glucose for providing energy to the brain in the starvation state (Cahill 2006).\u0026nbsp; Interference with ketogenesis, for example by PPAR\u0026alpha; inhibition, has been demonstrated to inhibit \u0026beta;-hydroxybutyrate production (measured in serum) during fasting events in mice (Badman et al 2007, Potthoff 2009).\u0026nbsp; Related to this observation, PPAR\u0026alpha;-knockout mice reached exhaustion sooner than wild types in an exercise challenge which corresponded with significantly decreased \u0026beta;-hydroxybutyrate in serum indicating hypoketonemia in PPAR\u0026alpha;-knockout mice versus wild types (Muoio et al 2002).\u0026nbsp; Under normal conditions, activated ketogenesis occurring during fasting events is rapidly deactivated when blood glucose concentrations increase to normal levels and resultant elevated circulating ketone bodies are reduced correspondingly (Cahill 2006).\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eKey Event 6: Catabolism of Muscle Protein, Increase\u003c/strong\u003e\u003cstrong\u003ed \u0026nbsp;- \u003c/strong\u003eAfter two to three days of fasting in humans, dietary glucose has been long-since expended and contribution to blood glucose from glycogen metabolism is reduced to zero (Cahill 2006).\u0026nbsp; At this point, about two fifths of fatty acid metabolism in the whole body is dedicated to hepatic ketogenesis, largely in support of the energy demands of the brain, however the brain is still significantly supported by glucose derived from gluconeogenesis (Cahill 2006).\u0026nbsp; As fatty acid stores are depleted, gluconeogenesis from other substrates becomes increasingly important including muscle protein catabolism \u003cem\u003ein situ\u003c/em\u003e for supporting muscle function as well as releasing glutamine (Marliss et al 1971) and alanine (Felig et al 1970A) which can be recycled to glucose by gluconeogenesis in the kidney (Goodman et al 1966, Kashiwaya et al 1994, Cahill 2006). In prolonged starvation events, the catabolism of muscle protein for gluconeogenesis in order to support systemic energy needs results in loss of muscle mass which contributes to loss of overall body weight.\u0026nbsp; This loss is rapidly reversible upon input of alternative metabolic fuel for example by nutrient assimilation from feeding.\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eAdverse Outcome:\u0026nbsp; Loss of body weight\u003c/strong\u003e\u003cstrong\u003e - \u003c/strong\u003eIf caloric intake is less than caloric use over time, an individual will lose body weight.\u0026nbsp; Dynamic energy budget theory has provided useful insights on how organisms take up, assimilate and then allocate energy to various fundamental biological processes including maintenance, growth, development and reproduction (Nisbet et al 2000).\u0026nbsp; Regarding energy allocation, somatic maintenance must first be met before then growth may occur, followed by maturation and then finally, surplus energy is dedicated to reproduction (Nisbet et al 2000).\u0026nbsp; The influence of PPARalpha on systemic energy metabolism and energy homeostasis has been broadly established (see reviews by Kersten 2014, Evans et al 2004, Desvergne and Wahli 1999).\u0026nbsp; PPARalpha has been demonstrated to play a critical role in stimulating fatty acid oxidation and ketogenesis during fasting resulting in increased ketone body levels in plasma (Badman et al 2007, Kersten 2014) a response that is eliminated in PPARalpha knockout mice (Badman et al 2007, Sanderson et al 2010).\u0026nbsp;\u0026nbsp; Kersten et al (1999) and Badman et al (2007) demonstrated that PPARalpha-null mice were unable to actively mobilize fatty acid oxidation, and further, Kersten et al (1999) demonstrated that these mice were unable to meet energy demands during fasting and leading to hypoglycemia, hyperlipidemia, hypoketonemia and fatty liver.\u0026nbsp;\u0026nbsp; Observations from toxicological and toxicogenomic research have implicated nitrotoluenes as potential PPAR antagonists in birds (Rawat et al 2010), rats (Deng et al 2011) and mice (Wilbanks et al 2014), an effect that additionally corresponded with weight loss in rats (Wilbanks et al 2014) and body weight loss, loss of muscle mass and emaciation in birds (Quinn et al 2007).\u0026nbsp; These combined results indicate that inhibition of PPARalpha signaling and the resultant decrease in fatty acid oxidation and ketogenesis can detrimentally impair systemic energy budgets leading to starvation-like effects and resultant weight loss.\u0026nbsp; In the absence of PPARalpha knockout, and upon removal of PPARalpha antagonist dosing, normal bioenergetic physiology can potentially be attained.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003e\u0026quot;\u0026#39;Biological Plausibility\u0026quot;\u0026#39;\u003c/p\u003e\r\n\r\n\u003cp\u003eBinding of molecules to peroxisome proliferator-activated receptor \u0026alpha; (PPAR\u0026alpha;) can cause either agonistic (i.e. GW409544) or antagonistic (i.e. GW6471) signaling depending on molecular structure (Xu et al 2001, Xu et al 2002).\u0026nbsp; A well described mode of antatonistic binding by GW6471 demonstrates that the molecule can bind to the PPAR\u0026alpha; ligand binding domain causing conformational changes that induce increased affinity to co-repressors which decrease PPAR\u0026alpha; nuclear signaling (Xu et al 2002) representing the MIE for this AOP. \u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eThe transcription co-repressors, \u003cu\u003es\u003c/u\u003eilencing \u003cu\u003em\u003c/u\u003eediator for \u003cu\u003er\u003c/u\u003eetinoid and \u003cu\u003et\u003c/u\u003ehyroid hormone receptors (SMRT) and \u003cu\u003en\u003c/u\u003euclear receptor \u003cu\u003eco-r\u003c/u\u003eepressor (N-CoR) have been observed to compete with transcriptional co-activators for binding to nuclear receptors (including PPAR\u0026alpha;) thus suppressing basal transcriptional activity (Nagy et al 1999, Xu et al 2002).\u0026nbsp; Regarding the KE1, the binding of co-repressors such as the SMRT and N-CoR to PPAR\u0026alpha; is reinforced by the MIE, which blocks the AF-2 helix from adopting the active conformation, as demonstrated in x-ray crystallography results presented in Xu et al (2002).\u0026nbsp; Thus, molecules that bind to PPAR\u0026alpha; that can enhance co-repressor binding act as PPAR\u0026alpha; antagonists.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eGiven that PPAR\u0026alpha; trans-activation induces catabolism of fatty acids, this signaling pathway has been broadly demonstrated to play a key role in energy homeostasis (Kersten 2014, Evans et al 2004, Desvergne and Wahli 1999).\u0026nbsp; In fact, PPAR\u0026alpha; regulates expression of genes encoding nearly every enzymatic step of fatty acid catabolism including fatty acid uptake into cells, fatty acid activation to acyl-CoAs, the release of cellular energy from fatty acids through the oxidative breakdown of acyl-CoAs to acetyl-CoA, and in starvation conditions, the repackaging of Acetyl-CoA substrates into ketone bodies (Kersten 2014, Desvergne and Wahli 1999, Evans et al 2004, Sengupta et al 2010).\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eA large body of research demonstrated that PPAR\u0026alpha; nuclear signaling directly controls transcriptional expression for genes catalyzing peroxisomal beta-oxidation of very long chain fatty acids (\u0026gt;20C), mitochondrial beta-oxidation of short, medium and long chain fatty acids (\u0026lt;20C), and ketogenesis (as reviewed in Kersten 2014, Evans et al 2004, Desvergne and Wahli 1999, Sanderson et al 2010, McMullen et al 2014, Rakhshandehroo et al 2009).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003ePeroxisomal beta-oxidation reactions shorten very long chain fatty acids from dietary sources releasing acetyl-CoA subunits (a primary metabolic fuel source) and shortened-chain fatty acids that can subsequently be catabolized by mitochondrial fatty acid beta oxidation reaction (as reviewed in Kersten et al 2014 and Desvergne and Wahli 1999).\u0026nbsp; Fatty acids shortened via peroxisomal beta-oxidation as well as fatty acids released from adipose tissue stores can be catabolized in mitochondrial beta-oxidation reactions to acetyl-CoA, NADH and ATP (Aoyama et al 1998).\u0026nbsp; Within the mitochondria, the acetyl-CoA substrates can be used to maximize ATP production through full substrate oxidation via the citric acid cycle followed by oxidative phosphorylation by the electron transport chain (Nelson and Cox 2000A, Desvergne and Wahli 1999).\u0026nbsp; This demonstrates the importance of PPAR\u0026alpha; signaling for inducing cellular energy release from fatty acids.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eBlocking PPAR\u0026alpha; signaling has been shown to inhibit expression of transcripts / enzymes involved in both peroxisomal and mitochondrial beta-oxidation causing impaired fatty acid catabolism, fatty acid accumulation in the liver and impaired cellular energy state during fasting events (Badman et al 2007, Kersten et al 1999).\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eDuring periods of fasting, acetyl-CoA generated during either peroxisomal or mitochondrial beta-oxidation of fatty acids in the liver can each contribute to ketogenesis (Kersten 2014, Sengupta 2010).\u0026nbsp; The liver represents a key organ involved in systemic energy distribution given its ability to synthesize glucose and catalyze the formation of ketone bodies, especially \u0026beta;-hydroxybutyrate, via ketogenesis (Cahil 2006, Kersten 2014).\u0026nbsp; \u0026beta;-hydroxybutyrate is especially important for the metabolic energy needs of the brain which is unable to utilize fatty acids for cellular energy production (Owen 2005, Kersten 2014) as well as supporting general systemic energy homeostasis in fasting events (Cahil 2006, Evans et al 2004).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eNot only does PPAR\u0026alpha; signaling stimulate the release of cellular energy from fatty acids, it also regulates the transcription of enzymes that catalyze the repackaging of that cellular energy to ketone bodies via ketogenesis (Sengupta et al 2010, Desvergne and Wahli 1999).\u0026nbsp; Inhibition of PPAR\u0026alpha; signaling has been demonstrated to inhibit transcriptional expression of genes that catalyze ketogenesis as well as ketone body production (Badman et al 2007, Potthoff 2009, Sengupta 2010) affecting circulating levels of ketone bodies for systemic use.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eKersten et al (1999) demonstrated that PPAR\u0026alpha; is induced in fasted mice mobilizing the oxidation of fatty acids for energy production.\u0026nbsp; In that study, PPAR\u0026alpha;-null mice did not actively induce fatty acid oxidation or ketogenesis leaving the mice unable to meet energy demands during fasting and leading to hypoglycemia, hyperlipidemia, hypoketonemia and fatty liver. \u0026nbsp;In such energy deficits, muscle protein catabolism is induced where the amino acids glutamine and alanine serve as substrates for gluconeogenesis in the kidney to supplement cellular energy production / distribution (Cahill 2006, Marliss et al 1971, Felig et al 1970A, Goodman et al 1966, Kashiwaya et al 1994).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003ePPAR\u0026alpha; knockout resulted in important organism-level responses including quicker onset of exhaustion compared to wild type mice in exercise trials where PPAR\u0026alpha;-K/Os exhibited hypoketonemia (Muoio et al 2002).\u0026nbsp; \u0026nbsp;Additionally, animals exposed to pollutants (nitrotoluenes) that act as partial PPAR\u0026alpha; antagonists had decreased exercise endurance (Wilbanks et al 2014), showed body weight loss (Wilbanks et al 2014, Quinn et al 2007) and displayed loss of muscle mass (Quinn et al 2007).\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eIn general, if caloric intake is less than caloric use over time, an individual will lose body weight.\u0026nbsp; This is a basic principle in human dieting as well as an important principle related to individual health and ecological fitness of animal populations.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eDynamic energy budget theory has provided useful insights on how organisms take up, assimilate and then allocate energy to various fundamental biological processes including maintenance, growth, development and reproduction (Nisbet et al 2000).\u0026nbsp; Regarding energy allocation, somatic maintenance must first be met before then growth may occur, followed by maturation and then finally, surplus energy is dedicated to reproduction (Nisbet et al 2000).\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eAs an example of the importance of energy allocation to ecological fitness, a review by Martin et al (1987) demonstrated that energy availability (availability of food) was the predominant limiting factor in reproductive success and survival for both young and parents in a broad life history review for bird species.\u0026nbsp; This is a likely scenario for many organisms.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026quot;\u0026#39;Concordance of dose-response relationships:\u0026quot;\u0026#39;\u003c/p\u003e\r\n\r\n\u003cp\u003eDose-response relationships have been developed for GW6471 and the relative binding of PPAR\u0026alpha; co-repressors and co-activators to the PPAR\u0026alpha; nuclear signaling complex where the proportion of co-repressors increases dramatically with increasing GW6471 concentration (Xu et al 2002).\u0026nbsp; Correspondingly, the relative activity of PPAR\u0026alpha; decreased to zero with increasing GW6471 concentrations (Xu et al 2002).\u0026nbsp; Additionally, recent observations of PPAR\u0026alpha; antagonism by nitrotoluenes have demonstrated dose-response relationships for PPAR\u0026alpha; nuclear signaling inhibition in human \u003cem\u003ein vitro\u003c/em\u003e investigations which corresponded with dose-responsive decreases in transcriptional expression of genes involved in lipid metabolism pathways (Wilbanks et al 2014, Gust et al 2015).\u0026nbsp; These results corresponded with a dose-responsive relationship where increasing nitrotoluene dose caused decreased muscle mass, decreased body weight and increased emaciation in chronic dosing studies (Quinn et al 2007).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026quot;\u0026#39;Temporal concordance among the key events and adverse effect:\u0026quot;\u0026#39;\u003c/p\u003e\r\n\r\n\u003cp\u003eCo-repressor binding was observed prior to inhibition of PPAR\u0026alpha; signaling (Xu et al 2002).\u0026nbsp; PPAR\u0026alpha; knock out nullifies downstream expression of transcripts for genes involved in peroxisomal beta-oxidation of fatty acids, mitochondrial beta-oxidation of fatty acids, and ketogenesis pathways relative to wild types (Kersten et al 2014).\u0026nbsp; Peroxisomal beta-oxidation of very long chain fatty acids into long chain fatty acids occurs prior to import into mitochondria and progression of mitochondrial beta-oxidation (Lazarow 1978, Kersten 2014).\u0026nbsp; Mitochondrial beta-oxidation of long chain fatty acids occurs prior to generation of ketone bodies via ketogenesis (Sengupta et al 2010, Badman et al 2007).\u0026nbsp; Ketogenesis occurs prior to increases in circulating ketone bodies (Sengupta et al 2010, Badman et al 2007, Cahill 2006).\u0026nbsp; Increases in circulating ketone bodies can be observed prior to loss of muscle mass to muscle-protein catabolism given that this linkage is not directly connected.\u0026nbsp; Muscle protein catabolism derives amino acids that are recycled to glucose via renal gluconeogenesis (Goodman et al 1966, Kashiwaya et al 1994, Cahill 2006).\u0026nbsp; Catabolism of muscle protein occurs prior to body weight loss (Quinn et al 2007).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026quot;\u0026#39;Consistency:\u0026quot;\u0026#39;\u003c/p\u003e\r\n\r\n\u003cp\u003eThe transcription co-repressors, \u003cu\u003es\u003c/u\u003eilencing \u003cu\u003em\u003c/u\u003eediator for \u003cu\u003er\u003c/u\u003eetinoid and \u003cu\u003et\u003c/u\u003ehyroid hormone receptors (SMRT) and \u003cu\u003en\u003c/u\u003euclear receptor \u003cu\u003eco-r\u003c/u\u003eepressor (N-CoR) competition with transcriptional co-activators for binding to nuclear receptors (including PPAR\u0026alpha;) has been observed in humans as well as yeast (Nagy et al 1999) suggesting broad taxonomic applicability for this MIE.\u0026nbsp; The evidence of PPAR\u0026alpha; as a regulator of fatty acid metabolism is well described in the literature (for example, Kersten 2014, Evans 2004, Desvergne and Wahili 1999), and is consistent across many species including human, mouse, rat, Northern bobwhite, fathead minnow and carp (Kersten et al 1999, Kersten 2014, Wintz et al 2006, Gust et al 2015, Deng et al 2011, Wilbanks et al 2014, Xu and Jing, 2012).\u0026nbsp; Inhibition of PPAR\u0026alpha; via gene knockout or treatment with PPAR\u0026alpha; antagonist consistently results in deceased fatty acid metabolism with indicators of increased serum triglycerides, fatty livers and steatosis (Kersten 2014, Evans 2004, Desvergne and Wahili 1999, Kersten et al 1999, Wintz et al 2006, Deng et al 2011).\u0026nbsp; Given PPAR\u0026alpha;\u0026rsquo;s central role in systemic energy metabolism, studies of PPAR\u0026alpha; antagonism have shown decreased potential for sustaining energy needs of the organism (Kersten et al 1999) leading to decreased exercise performance (Muoio et al 2002, Wilbanks et al 2014) and weight loss (Wilbanks et al 2014, Quinn et al 2007).\u0026nbsp; Research thus far suggest that the PPAR\u0026alpha; transcriptional regulation pathway as well as the metabolic pathways for which PPAR\u0026alpha; acts as a regulator indicates that the progression of key events through to the adverse outcome will tend to be evolutionarily conserved for within mammals and likely across animal phyla.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026quot;\u0026#39;Uncertainties, inconsistencies, and data gaps:\u0026quot;\u0026#39;\u003c/p\u003e\r\n\r\n\u003cp\u003eA critical data gap regarding this AOP is an absence of studies that have investigated the effects null mutants for ketogenesis on the physiology and individual performance during long term starvation relative to wild type individuals.\u0026nbsp; Additionally, knowledge about feedback mechanisms between ketogenesis vs gluconeogenesis would be beneficial for interpreting systemic energy metabolism.\u0026nbsp; Regarding the antagonistic action of nitrotoluenes on PPARalpha nuclear signaling (Wilbanks et al 2014, Gust et al 2015), receptor-binding assays would be beneficial to determine if this class of chemicals is binding the SMRT and N-CoR co-repressors, similar to the antagonistic action of GW6471 (Xu et al 2002).\u003c/p\u003e\r\n","quantitative_considerations":"\u003cp\u003eGiven the complex nature of PPARalpha\u0026rsquo;s functioning within a multi-subunit transcription factor regulating the transcriptional expression of a multitude of genes that facilitate lipid metabolism, to our knowledge, the relationship between PPARalpha signaling and individual gene expression has not yet been quantitatively modeled.\u0026nbsp; However, the gene regulatory networks structure is well established (KEGG Pathway, map03320) and numerous empirical observations of the positive relationship between PPARalpha signaling with transcript expression and downstream metabolic pathways (Kersten 2014, Desvergne and Wahli 1999), there is opportunity to develop a quantitative gene signaling model for this system.\u0026nbsp; For peroxisomal and mitochondrial fatty acid beta-oxidation pathways and ketogenesis, a variety of enzyme kinetics information is available for modeling (see reviews by Kersten 2014, Desvergne and Wahli 1999) as well as basic knowledge of the reaction stoichiometry of each metabolic reactions that can contribute to metabolic energy substrates for systemic use.\u0026nbsp; Resultant models should be integrated with the work of Kashiwaya et al (1994) who have developed a detailed quantitative model for the metabolic flux of glucose including the influence of ketone bodies and insulin action on the dynamics of glycolysis versus gluconeogenesis.\u0026nbsp; Dynamic energy budget (DEB) models (Nisbet et al 2000) have strong utility for integrating the dynamics of energy input and allocation to organismal processes of importance for characterizing/predicting the condition of the individual (ie. growth and maturation) as well as population-level responses (ie. allocation of energy to reproduction).\u0026nbsp; DEB modeling has great potential for integrating suborganismal processes into individual and population level outcomes (Ananthasubramaniam et al 2015) and could serve to integrate data from dose-responsive relationships among PPARalpha antagonistic nitrotoluenes and fatty acid metabolism, muscle loss and body weight loss (Rawat et al 2010, Deng et al 2011, Wilbanks et al 2014, Quinn et al 2007, Xu and Jin 2012) thus supporting development of a semi-quantitative or quantitative AOP.\u003c/p\u003e\r\n","optional_considerations":"\u003ch2\u003eThe present AOP may require additional conditions to be fully manifested.\u0026nbsp; \u0026nbsp; The risk for this AOP is expected to be exacerbated during fasting, starvation and/or sub-optimal nutrition where interference with PPAR\u0026alpha; signaling is likely to contribute synergistically toward decreased exercise performance and increased body-weight loss.\u0026nbsp;\u003c/h2\u003e\r\n","references":"\u003cp\u003eAnanthasubramaniam B, McCauley E, Gust KA, Kennedy AJ, Muller EB, Perkins EJ, Nisbet RM: Relating suborganismal processes to ecotoxicological and population level endpoints using a bioenergetic model. \u003cem\u003eEcol Appl\u003c/em\u003e 2015, 25(6):1691-1710.\u003c/p\u003e\r\n\r\n\u003cp\u003eAoyama, T., Peters, J.M., Iritani, N., Nakajima, T., Furihata, K., Hashimoto, T., et al., 1998. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). Journal of Biological Chemistry 273:5678e5684.\u003c/p\u003e\r\n\r\n\u003cp\u003eBadman MK, Pissios P, Kennedy AR, Koukos G, Flier JS, Maratos-Flier E: Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states. \u003cem\u003eCell metabolism \u003c/em\u003e2007, 5(6):426-437.\u003c/p\u003e\r\n\r\n\u003cp\u003eBrandt JM, Djouadi F, Kelly DP: Fatty acids activate transcription of the muscle carnitine palmitoyltransferase I gene in cardiac myocytes via the peroxisome proliferator-activated receptor alpha. \u003cem\u003eJ Biol Chem \u003c/em\u003e1998, 273(37):23786-23792.\u003c/p\u003e\r\n\r\n\u003cp\u003eCahill Jr., G.F., 2006. Fuel metabolism in starvation. Annual Review of Nutrition 26:1e22.\u003c/p\u003e\r\n\r\n\u003cp\u003eCollier\u0026nbsp;ZA, Gust KA, Gonzalez-Morales B, Linkov I, Perkins EJ (Eligible for publication, pending revisions) A Weight of Evidence Assessment Approach for Adverse Outcome Pathways. Regul Toxicol Pharm.\u003c/p\u003e\r\n\r\n\u003cp\u003eDeng Y, Johnson DR, Guan X, Ang CY, Ai J, Perkins EJ (2010) In vitro gene regulatory networks predict in vivo function of liver. BMC Systems Biology 4:153.\u003c/p\u003e\r\n\r\n\u003cp\u003eDeng, Y., Meyer, S. A., Guan, X., Escalon, B. L., Ai, J.,Wilbanks, M.S., Welti, R., Garcia-Reyero, N. and Perkins, E. J. (2011) Analysis of common and specific mechanisms of liver function affected by nitrotoluene compounds. PLoS One 6, e14662.\u003c/p\u003e\r\n\r\n\u003cp\u003eDesvergne B, Wahli W (1999) Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews 20(5): 649-688.\u003c/p\u003e\r\n\r\n\u003cp\u003eEvans RM, Barish GD, Wang YX: PPARs and the complex journey to obesity. Nat Med 2004, 10(4):355-361.\u003c/p\u003e\r\n\r\n\u003cp\u003eFeige, J.N., Gerber, A., Casals-Casas, C., Yang, Q., Winkler, C., Bedu, E., Bueno, M., Gelman, L., Auwerx, J., Gonzalez, F.J., Desvergne, B., 2010. The pollutant diethylhexyl phthalate regulates hepatic energy metabolism via species-specific PPARalpha-dependent mechanisms. \u003cem\u003eEnviron. Health Perspect.\u003c/em\u003e 118, 234-241.\u003c/p\u003e\r\n\r\n\u003cp\u003eFelig P, Pozefsky T, Marliss E, Cahill GF, Jr.: Alanine: key role in gluconeogenesis. \u003cem\u003eScience \u003c/em\u003e1970A, 167(3920):1003-1004.\u003c/p\u003e\r\n\r\n\u003cp\u003eFelig P, Marliss E, Pozefsky T, Cahill GF, Jr.: Amino acid metabolism in the regulation of gluconeogenesis in man. \u003cem\u003eAm J Clin Nutr \u003c/em\u003e1970B, 23(7):986-992.\u003c/p\u003e\r\n\r\n\u003cp\u003eGerich JE, Meyer C, Woerle HJ, Stumvoll M: Renal gluconeogenesis: its importance in human glucose homeostasis. \u003cem\u003eDiabetes Care \u003c/em\u003e2001, 24(2):382-391.\u003c/p\u003e\r\n\r\n\u003cp\u003eGoodman AD, Fuisz RE, Cahill GF: Renal gluconeogenesis in acidosis, alkalosis, and potassium deficiency: its possible role in regulation of renal ammonia production. \u003cem\u003eJ Clin Invest\u003c/em\u003e 1966, 45(4):612-619.\u003c/p\u003e\r\n\r\n\u003cp\u003eGust KA, Nanduri B, Rawat A, Wilbanks MS, Ang CY, Johnson DR, Pendarvis K, Chen X, Quinn Jr. MJ, Johnson MS, Burgess SC, Perkins EJ (2015) Systems Toxicology Identifies Mechanistic Impacts of 2-amino-4,6-dinitrotoluene (2A-DNT) Exposure in Northern Bobwhite. BMC Genomics. In Press.\u003c/p\u003e\r\n\r\n\u003cp\u003eIde T, Shimano H, Yoshikawa T, Yahagi N, Amemiya-Kudo M, Matsuzaka T, Nakakuki M, Yatoh S, Iizuka Y, Tomita S, et al (2003) Cross-talk between peroxisome proliferator-activated receptor (PPAR) alpha and liver X receptor (LXR) in nutritional regulation of fatty acid metabolism. II. LXRs suppress lipid degradation gene promoters through inhibition of PPAR signaling. Mol Endocrinol 17(7):1255-1267.\u003c/p\u003e\r\n\r\n\u003cp\u003eJanssen, A.W., Betzel, B., Stoopen, G., Berends, F.J., Janssen, I.M., Peijnenburg, A.A., Kersten, S., 2015. The impact of PPARalpha activation on whole genome gene expression in human precision cut liver slices. \u003cem\u003eBMC Genomics\u003c/em\u003e 16, 760.Kashiwaya Y, Sato K, Tsuchiya N, Thomas S, Fell DA, Veech RL, Passonneau JV: Control of glucose utilization in working perfused rat heart. \u003cem\u003eJ Biol Chem \u003c/em\u003e1994, 269(41):25502-25514.\u003c/p\u003e\r\n\r\n\u003cp\u003eKersten S.\u0026nbsp; 2014. Integrated physiology and systems biology of PPARalpha. Molecular Metabolism 2014, 3(4):354-371.\u003c/p\u003e\r\n\r\n\u003cp\u003eKersten S, Seydoux J, Peters JM, Gonzalez FJ, Desvergne B, Wahli W: Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting. J Clin Invest 1999, 103(11):1489-1498.\u003c/p\u003e\r\n\r\n\u003cp\u003eLazarow PB: Rat liver peroxisomes catalyze the beta oxidation of fatty acids. J Biol Chem 1978, 253(5):1522-1528.\u003c/p\u003e\r\n\r\n\u003cp\u003eLe May et al., 2000. Reduced hepatic fatty acid oxidation in fasting PPARK null mice is due to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression. FEBS Lett. 475: 163-166.\u003c/p\u003e\r\n\r\n\u003cp\u003eLefebvre P, Chinetti G, Fruchart J-C, Staels B (2006) Sorting out the roles of PPAR\u0026alpha; in energy metabolism and vascular homeostasis. Journal of Clinical Investigation, 116(3), 571\u0026ndash;580.\u003c/p\u003e\r\n\r\n\u003cp\u003eLiu, M.H., Li, J., Shen, P., Husna, B., Tai, E.S., Yong, E.L., 2008. A natural polymorphism in peroxisome proliferator-activated receptor-alpha hinge region attenuates transcription due to defective release of nuclear receptor corepressor from chromatin. \u003cem\u003eMol. Endocrinol.\u003c/em\u003e 22, 1078-1092.\u003c/p\u003e\r\n\r\n\u003cp\u003eMarliss EB, Aoki TT, Pozefsky T, Most AS, Cahill GF: Muscle and splanchnic glutamine and glutamate metabolism in postabsorptive and starved man. \u003cem\u003eJ Clin Invest \u003c/em\u003e1971, 50(4):814-817.\u003c/p\u003e\r\n\r\n\u003cp\u003eMartin TE: Food as a limit on breeding birds: a life-history perspective. \u003cem\u003eAnnu Rev Ecol Syst \u003c/em\u003e1987:453-487.\u003c/p\u003e\r\n\r\n\u003cp\u003eMascar\u0026oacute; C, Acosta E, Ortiz JA, Marrero PF, Hegardt FG, Haro D: Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor. \u003cem\u003eJ Biol Chem \u003c/em\u003e1998, 273(15):8560-8563.\u003c/p\u003e\r\n\r\n\u003cp\u003eMcMullen, P.D., Bhattacharya, S., Woods, C.G., Sun, B., Yarborough, K., Ross, S.M., Miller, M.E., McBride, M.T., LeCluyse, E.L., Clewell, R.A., Andersen, M.E., 2014. A map of the PPARalpha transcription regulatory network for primary human hepatocytes. \u003cem\u003eChem. Biol. Interact.\u003c/em\u003e 209, 14-24.\u003c/p\u003e\r\n\r\n\u003cp\u003eMuoio, D.M., MacLean, P.S., Lang, D.B., Li, S., Houmard, J.A., Way, J.M., Winegar, D.A., Corton, J.C., Dohm, G.L., Kraus, W.E., 2002. Fatty acid homeostasis and induction of lipid regulatory genes in skeletal muscles of peroxisome proliferator-activated receptor (PPAR) alpha knock-out mice. Evidence for compensatory regulation by PPAR delta. J. Biol. Chem. 277, 26089-26097.\u003c/p\u003e\r\n\r\n\u003cp\u003eNagy L, Kao H-Y, Love JD, Li C, Banayo E, Gooch JT, Krishna V, Chatterjee K, Evans RM, Schwabe JWR: Mechanism of corepressor binding and release from nuclear hormone receptors. \u003cem\u003eGenes Dev \u003c/em\u003e1999, 13(24):3209-3216.\u003c/p\u003e\r\n\r\n\u003cp\u003eNelson DL, Cox MM 2000A.\u0026nbsp; The Citric Acid Cycle. \u003cem\u003eLehninger Principles of Biochemistry\u003c/em\u003e. 3\u003csup\u003erd\u003c/sup\u003e Edition.\u0026nbsp; Worth Publishers.\u0026nbsp; New York, NY. p567-592.\u003c/p\u003e\r\n\r\n\u003cp\u003eNelson DL, Cox MM 2000B.\u0026nbsp; Carbohydrate Biosynthesis. \u003cem\u003eLehninger Principles of Biochemistry\u003c/em\u003e. 3\u003csup\u003erd\u003c/sup\u003e Edition.\u0026nbsp; Worth Publishers.\u0026nbsp; New York, NY. p722-764.\u003c/p\u003e\r\n\r\n\u003cp\u003eNelson DL, Cox MM 2000C.\u0026nbsp; Lipid Biosynthesis. \u003cem\u003eLehninger Principles of Biochemistry\u003c/em\u003e. 3\u003csup\u003erd\u003c/sup\u003e Edition.\u0026nbsp; Worth Publishers.\u0026nbsp; New York, NY. p770-814.\u003c/p\u003e\r\n\r\n\u003cp\u003eNisbet R, Muller E, Lika K, Kooijman S: From molecules to ecosystems through dynamic energy budget models. \u003cem\u003eJ Anim Ecol \u003c/em\u003e2000, 69(6):913-926.\u003c/p\u003e\r\n\r\n\u003cp\u003eOwen OE: Ketone bodies as a fuel for the brain during starvation. Biochem Mol Biol Educ 2005, 33(4):246-251.\u003c/p\u003e\r\n\r\n\u003cp\u003ePotthoff MJ, Inagaki T, Satapati S, Ding X, He T, Goetz R, Mohammadi M, Finck BN, Mangelsdorf DJ, Kliewer SA\u003cem\u003e et al\u003c/em\u003e: FGF21 induces PGC-1\u0026alpha; and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response. \u003cem\u003eProceedings of the National Academy of Sciences \u003c/em\u003e2009, 106(26):10853-10858.\u003c/p\u003e\r\n\r\n\u003cp\u003eQuinn MJ Jr, Bazar MA, McFarland CA, Perkins EJ, Gust KA, Gogal Jr RM, Johnson MS (2007) Effects of subchronic exposure to 2,6-dinitrotoluene in the northern bobwhite (Colinus virginianus). Environmental Toxicology and Chemistry 26(10):2202-2207.\u003c/p\u003e\r\n\r\n\u003cp\u003eRakhshandehroo, M., Hooiveld, G., Muller, M., Kersten, S., 2009. Comparative analysis of gene regulation by the transcription factor PPARalpha between mouse and human. \u003cem\u003ePLoS One\u003c/em\u003e 4, e6796.\u003c/p\u003e\r\n\r\n\u003cp\u003eRawat A, Gust KA, Deng Y, Garcia-Reyero N, Quinn MJ, Jr., Johnson MS, Indest KJ, Elasri MO, Perkins EJ (2010) From raw materials to validated system: the construction of a genomic library and microarray to interpret systemic perturbations in Northern bobwhite. Physiological Genomics 42(2):219-235.\u003c/p\u003e\r\n\r\n\u003cp\u003eSanderson, L.M., Boekschoten, M.V., Desvergne, B., Muller, M., Kersten, S., 2010. Transcriptional profiling reveals divergent roles of PPARalpha and PPARbeta/delta in regulation of gene expression in mouse liver. Physiological Genomics 41:42e52.\u003c/p\u003e\r\n\r\n\u003cp\u003eSengupta S, Peterson TR, Laplante M, Oh S, Sabatini DM: mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. \u003cem\u003eNature \u003c/em\u003e2010, 468(7327):1100-1104.\u003c/p\u003e\r\n\r\n\u003cp\u003eWang YX: PPARs: diverse regulators in energy metabolism and metabolic diseases. Cell Res 2010, 20(2):124-137.\u003c/p\u003e\r\n\r\n\u003cp\u003eWilbanks, M., Gust, K.A., Atwa, S., Sunesara, I., Johnson, D., Ang, C.Y., Meyer., S.A., and Perkins, E.J. 2014. Validation of a genomics-based hypothetical adverse outcome pathway: 2,4-dinitrotoluene perturbs PPAR signaling thus impairing energy metabolism and exercise endurance. Toxicological Sciences. 141(1):44-58.\u003c/p\u003e\r\n\r\n\u003cp\u003eWintz, H., Yoo, L. J., Loguinov, A., Wu, Y., Steevens, J. A., Holland, R. D., Beger, R. D., Perkins, E. J., Hughes, O. and Vulpe, C. D. (2006) Gene expression profiles in fathead minnow exposed to 2,4-DNT correlation with toxicity in mammals. Toxicol. Sci. 94, 71\u0026ndash;82.\u003c/p\u003e\r\n\r\n\u003cp\u003eWolfrum C, Borrmann CM, Borchers T, Spener F (2001) Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha - and gamma-mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. Proc Natl Acad Sci USA 98(5):2323-2328.\u003c/p\u003e\r\n\r\n\u003cp\u003eXu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL, Oplinger JA, Kliewer SA, Gampe RT, McKee DD\u003cem\u003e et al\u003c/em\u003e 2001 \u0026nbsp;Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors. \u003cem\u003eProceedings of the National Academy of Sciences \u003c/em\u003e\u0026nbsp;98(24):13919-13924.\u003c/p\u003e\r\n\r\n\u003cp\u003eXu HE, Stanley TB, Montana VG, Lambert MH, Shearer BG, Cobb JE, McKee DD, Galardi CM, Plunket KD, Nolte RT\u003cem\u003e et al\u003c/em\u003e: Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPAR[alpha]. \u003cem\u003eNature \u003c/em\u003e2002, 415(6873):813-817.\u003c/p\u003e\r\n\r\n\u003cp\u003eXu, J. and Jing, N. (2012) Effects of 2,4-dinitrotoluene exposure on enzyme activity, energy reserves and condition factors in common carp (Cyprinus carpio). J. Hazard. Mater. 203\u0026ndash;204, 299\u0026ndash;307.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003eThe majority of the evidence described in this AOP are derived for either human (mostly in vitro) or mice (in vivo and in vitro) studies.\u0026nbsp; There are recognized differences between mouse and human PPAR\u0026alpha; signaling and responses from the literature, however, for our specific KEs, the responses among species are relatively well conserved.\u0026nbsp; Therefore, we have reasonable confidence that the AOP provides reliable confidence for human health assessment.\u0026nbsp; The AOP also has the potential to support ecotoxicological assessment if there is reasonable confidence that the KEs are conserved in the species of interest.\u0026nbsp; The risk for this AOP is expected to be exacerbated during fasting, starvation and/or sub-optimal nutrition where interference with PPAR\u0026alpha; signaling is likely to contribute synergistically toward decreased exercise performance in the short-term and drive body-weight loss in long-term exposures.\u0026nbsp; The molecular responses from the MIE through KE4 are very well characterized in the literature for human and mouse.\u0026nbsp; KE5 and KE6 have fairly strong support from the literature, however the KER between them, especially stemming back to the MIE remains the largest data gap within the AOP.\u0026nbsp; Finally, the connection between KE6 and the AO is intuitive and well established in the literature.\u0026nbsp; Overall, the AOP is biologically plausible with logical order where AO is likely to be exacerbated when nutrition is suboptimal.\u003c/p\u003e\r\n\r\n\u003cp\u003eRegarding the use of the AOP for chemical safety assessment, the AOP should have relevance for any chemical observed to inhibit PPAR\u0026alpha; signaling. \u0026nbsp;Additionally, the manifestation and severity of the AO is expected to occur predominantly in chronic exposures, especially in nutritionally stressed populations. \u0026nbsp;There is much left to learn about what chemical structures are likely to antagonistically bind to PPAR\u0026alpha; before quantitative structure-activity relationships (QSARs) can be developed to predict binding / antagonistic effects. \u0026nbsp;\u003c/p\u003e\r\n","background":"","user_defined_mie":"998: Binding of antagonist, PPAR alpha","user_defined_ao":"864: Decreased, Body Weight","oecd_project":"2.3","oecd_status_id":1,"graphical_representation_image_uid":"2018/06/11/1zmymvvz5m_AOP_Diagram.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2023-02-13T18:04:26.000-05:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":5,"handbook_id":1,"project_129":false},{"id":7,"title":"Aromatase (Cyp19a1) reduction leading to impaired fertility in adult female","short_name":"Aromatase (Cyp19a1) reduction leading to reproductive toxicity","corresponding_author_id":109,"abstract":"\u003cp\u003eThis AOP links activation of the Peroxisome Proliferator Activated Receptor\u0026gamma; (PPAR\u0026gamma;) to reproductive toxicity in adult female. The development of this AOP relies on evidence collected from rodent models and incorporates human mechanistic and epidemiological data. The PPAR\u0026gamma; is a ligand-activated transcription factor that belongs to the nuclear receptor family, which also includes the steroid and thyroid hormone receptors. Interest in PPAR\u0026gamma; action as a mechanistic basis for effects on the reproductive system arises from the demonstrated relationships between activation of this receptor and impairment of the steroidogenesis leading to reproductive toxicity in rodents. PPARs play important roles in the metabolic regulation of lipids, of which cholesterol, in particular being a precursor of steroid hormones, makes the link between lipid metabolism to effects on reproduction. The key events in the pathway comprise the activation of PPAR\u0026gamma;, followed by the disruption of the hormonal balance which leads to irregularities of the ovarian cycle that may further be cause of impaired fertility. The PPAR\u0026gamma;-initiated AOP to rodent female reproductive toxicity is a first step for structuring current knowledge about a mode of action which is neither ER-mediated nor via direct aromatase inhibition. In the current form the pathway lays a strong basis for linking an endocrine mode of action with an apical endpoint, prerequisite requirement for the identification of endocrine disrupting chemicals. This AOP is complemented with a structured data collection which will serve as the basis for further quantitative development of the pathway.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:15.000-05:00","updated_at":"2023-04-29T16:02:54.000-04:00","status_id":1,"authors":"\u003cp\u003eMalgorzata Nepelska, Elise Grignard, Sharon Munn,\u003c/p\u003e\r\n\r\n\u003cp\u003eSystems Toxicology Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Via E. Fermi 2749, I-21027 Ispra, Varese, Italy\u003c/p\u003e\r\n\r\n\u003cp\u003eCorresponding author: sharon.munn@ec.europa.eu; elise.grignard@ec.europa.eu\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003eThis AOP is relevant for mature females for details see reviews [(Kay, Chambers, and Foster 2013), (Froment et al. 2006), (Peraza et al. 2006), (Latini et al. 2008), (Martino-Andrade and Chahoud 2010), (Lyche et al. 2009), (Lovekamp-Swan and Davis 2003)].\u003c/p\u003e\r\n\r\n\u003cp\u003eThe experimental support for the pathway is based on rodent models and other mammals (pig, sheep) including human mechanistic and epidemiological data. The experimental animal data are assumed relevant for consideration of human risk.\u003c/p\u003e\r\n\r\n\u003cp\u003eThis AOP applies to females only for details see reviews [(Kay, Chambers, and Foster 2013), (Froment et al. 2006), (Peraza et al. 2006), (Latini et al. 2008), (Martino-Andrade and Chahoud 2010), (Lyche et al. 2009), (Lovekamp-Swan and Davis 2003)].\u003c/p\u003e\r\n","key_event_essentiality":"\u003ctable border=\"1\" style=\"border-collapse:collapse; font-size:75%\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKRs WoE\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eEssentiality - KEs\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003elevel of confidence\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003ca href=\"/wiki/index.php/Event:228\" title=\"Event:228\"\u003ePPAR gamma, Activation \u003c/a\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePPAR\u0026gamma; activation was found to indirectly alter the expression of aromatase\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eweak\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003ca href=\"/wiki/index.php/Event:408\" title=\"Event:408\"\u003eAromatase (Cyp19a1), reduction in ovarian granulosa cells\u003c/a\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eAromatase is the cytochrome P450 enzyme complex responsible for the conversion of androgens to estrogens during steroidogenesis which is a key reaction in the sex differentiation in vertebrates. Alterations in the amount of aromatase present or in the catalytic activity of the enzyme will alter the levels of estrogens in tissues and dramatically disrupt estrogen hormone action.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003emoderate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u003ca href=\"/wiki/index.php/Event:3\" title=\"Event:3\"\u003e17beta-estradiol synthesis by ovarian granulosa cells\u003c/a\u003e\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWhile both brain and adrenal tissue are capable of synthesizing estradiol, the gonads are generally considered the major source of circulating estrogens in vertebrates, including fish (Norris 2007). Consequently, if estradiol synthesis by ovarian granulosa cells is reduced, plasma E2 concentrations would be expected to decrease unless there are concurrent reductions in the rate of E2 catabolism.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003estrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003ca href=\"/wiki/index.php/Event:219\" title=\"Event:219\"\u003ePlasma 17beta-estradiol concentrations, Reduction\u003c/a\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eEstrogens are crucial for female fertility, as proved by the severe reproductive defects observed when their synthesis is blocked.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003estrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003ca href=\"/wiki/index.php/Event:405\" title=\"Event:405\"\u003eovarian cycle irregularities\u003c/a\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eA sequential progression of interrelated physiological and behavioural cycles underlines the female reproductive function.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003estrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003ca href=\"/wiki/index.php/Event:406\" title=\"Event:406\"\u003eFertility, impaired\u003c/a\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eImpaired Fertility is the endpoint of reproductive toxicity\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003estrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n","weight_of_evidence_summary":"\u003ctable border=\"1\" style=\"border-collapse:collapse; font-size:75%\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKERs \u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eBiological plausibility\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003elevel of confidence\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eEmpirical Support\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003elevel of confidence\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eInconsistencies/Uncertainties\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eDose-response\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTemporality\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eIncidence\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePPAR\u0026gamma;, Activation =\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAromatase (Cyp19a1), reduction in ovarian granulosa cells\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eThere is functional relationship between PPAR\u0026gamma; activation and reduction in aromatase levels. Several mechanisms have been investigated; however there is no established consensus.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eModerate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEup occurs at lower dose than KEdown(dose response concordance)\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eoccurrence of the key events at similar dose and time point\u003c/li\u003e\r\n\t\t\t\t\u003cli\u003eSupport for solid temporal relationship is lacking\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWeak\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eLimited data, for details see KER pages\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAromatase (Cyp19a1), reduction in ovarian granulosa cells =\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003e17beta-estradiol synthesis by ovarian granulosa cells\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWithin the ovary, aromatase expression and activity is primarily localized in the granulosa cells. Therefore, changes in ovarian aromatase can generally be assumed to directly impact E2 synthesis by the granulosa cells.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eModerate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEup occurs at lower dose than KEdown(dose response concordance)\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eoccurrence of the key events at similar dose and time point\u003c/li\u003e\r\n\t\t\t\t\u003cli\u003eSupport for solid temporal relationship is lacking\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003emoderate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eLimited data\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003e17beta-estradiol synthesis by ovarian granulosa cells, Reduction =\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePlasma 17beta-estradiol concentrations\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eThe gonads are generally considered the major source of circulating estrogens, consequently, if estradiol synthesis by ovarian granulosa cells is reduced, plasma E2 concentrations would be expected to decrease.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eStrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEs occur at similar dose levels\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eoccurrence of the key events at similar dose Support for solid temporal relationship is lacking\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003emoderate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eLimited data\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePlasma 17beta-estradiol concentrations, Reduction =\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eovarian cycle irregularities \u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eAlterations in relationships among the hypothalamic, pituitary, and ovarian components of the reproductive axis can have marked effects on cyclicity. A toxicological insult to any one of these sites can disrupt the cycle and block ovulation.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eStrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEs occur at similar dose levels\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eoccurrence of the key events at similar dose and time point\u003c/li\u003e\r\n\t\t\t\t\u003cli\u003eSupport for solid temporal relationship is lacking\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003emoderate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eovarian cycle irregularities =\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFertility, impaired \u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eA sequential progression of interrelated physiological and behavioural cycles underlines the female\u0026#39;s fertility and successful production of offspring.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eStrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEs occur at similar dose levels\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eoccurrence of the key events at similar dose and with temporal relationship\u003c/li\u003e\r\n\t\t\t\t\u003cli\u003eSupport for solid temporal relationship is lacking.\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003emoderate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eTable 1 Weight of Evidence Summary. The underlying questions for the content of table: Dose-response: Does the empirical evidence support that a change in KEup leads to an appropriate change in KEdown?; Temporality: Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup \u0026gt; than that for KEdown?; Incidence: Is there higher incidence of KEup than of KEdown?; Inconsistencies/Uncertainties: Are there inconsistencies in empirical support across taxa, species and stressors that don\u0026rsquo;t align with expected pattern for hypothesized AOP?\u003c/p\u003e\r\n","quantitative_considerations":"","optional_considerations":"\u003cp\u003e1. The AOP describes a pathway which allows for the detection of sex steroid-related endocrine disrupting modes of action, with focus on the identification of substances which affect the reproductive system. In the current form the pathway lays a strong basis for linking endocrine mode of action with an apical endpoint, a prerequisite requirement for identification of endocrine disrupting chemicals (EDC). EDCs require specific evaluation under REACH (1907/2006, Registration, Evaluation, Authorisation and Restriction of Chemicals (EU, 2006)), the revised European plant protection product regulation 1107/2009 (EU, 2009) and use of biocidal products 528/2012 EC (EU, 2012).Amongst other agencies the US EPA is also giving particular attention to EDCs (EPA, 1998).\u003c/p\u003e\r\n\r\n\u003cp\u003e2. This AOP structurally represents current knowledge of the pathway from PPAR\u0026gamma; activation to impaired fertility that may provide a basis for development (and interpretation) of strategies for Integrated Approaches to Testing Assessment (IATA) to identify similar substances that may operate via the same pathway related to sex steroids disruption and effects on reproductive tract and fertility. This AOP forms the starting point on an AOP network mapping modes of action for endocrine disruption.\u003c/p\u003e\r\n\r\n\u003cp\u003e3. The AOP could inform the development of quantitative structure activity relationships, read-across models, and/or systems biology models to prioritize chemicals for further testing.\u003c/p\u003e\r\n","references":"\u003cp\u003eCui, Yongzhi, Keiko Miyoshi, Estefania Claudio, Ulrich K Siebenlist, Frank J Gonzalez, Jodi Flaws, Kay-Uwe Wagner, and Lothar Hennighausen. 2002. \u0026ldquo;Loss of the Peroxisome Proliferation-Activated Receptor Gamma (PPARgamma ) Does Not Affect Mammary Development and Propensity for Tumor Formation but Leads to Reduced Fertility.\u0026rdquo; The Journal of Biological Chemistry 277 (20) (May 17): 17830\u0026ndash;5. doi:10.1074/jbc.M200186200.\u003c/p\u003e\r\n\r\n\u003cp\u003eFan, WuQiang, Toshihiko Yanase, Hidetaka Morinaga, Yi-Ming Mu, Masatoshi Nomura, Taijiro Okabe, Kiminobu Goto, Nobuhiro Harada, and Hajime Nawata. 2005. \u0026ldquo;Activation of Peroxisome Proliferator-Activated Receptor-Gamma and Retinoid X Receptor Inhibits Aromatase Transcription via Nuclear Factor-kappaB.\u0026rdquo; Endocrinology 146 (1) (January): 85\u0026ndash;92. doi:10.1210/en.2004-1046.\u003c/p\u003e\r\n\r\n\u003cp\u003eFroment, P, F Gizard, D Defever, B Staels, J Dupont, and P Monget. 2006. \u0026ldquo;Peroxisome Proliferator-Activated Receptors in Reproductive Tissues: From Gametogenesis to Parturition.\u0026rdquo; The Journal of Endocrinology 189 (2) (May): 199\u0026ndash;209. doi:10.1677/joe.1.06667.\u003c/p\u003e\r\n\r\n\u003cp\u003eGasic, S, Y Bodenburg, M Nagamani, A Green, and R J Urban. 1998. \u0026ldquo;Troglitazone Inhibits Progesterone Production in Porcine Granulosa Cells.\u0026rdquo; Endocrinology 139 (12) (December): 4962\u0026ndash;6. doi:10.1210/endo.139.12.6385.\u003c/p\u003e\r\n\r\n\u003cp\u003eHerreros, Maria A, Teresa Encinas, Laura Torres-Rovira, Rosa A Garcia-Fernandez, Juana M Flores, Jose M Ros, and Antonio Gonzalez-Bulnes. 2013. \u0026ldquo;Exposure to the Endocrine Disruptor di(2-Ethylhexyl)phthalate Affects Female Reproductive Features by Altering Pulsatile LH Secretion.\u0026rdquo; Environmental Toxicology and Pharmacology 36 (3) (November): 1141\u0026ndash;9. doi:10.1016/j.etap.2013.09.020.\u003c/p\u003e\r\n\r\n\u003cp\u003eKay, Vanessa R, Christina Chambers, and Warren G Foster. 2013. \u0026ldquo;Reproductive and Developmental Effects of Phthalate Diesters in Females.\u0026rdquo; Critical Reviews in Toxicology 43 (3) (March): 200\u0026ndash;19. doi:10.3109/10408444.2013.766149.\u003c/p\u003e\r\n\r\n\u003cp\u003eKim, Jaeyeon, Marcey Sato, Quanxi Li, John P Lydon, Francesco J Demayo, Indrani C Bagchi, and Milan K Bagchi. 2008. \u0026ldquo;Peroxisome Proliferator-Activated Receptor Gamma Is a Target of Progesterone Regulation in the Preovulatory Follicles and Controls Ovulation in Mice.\u0026rdquo; Molecular and Cellular Biology 28 (5) (March): 1770\u0026ndash;82. doi:10.1128/MCB.01556-07.\u003c/p\u003e\r\n\r\n\u003cp\u003eKomar, C M, O Braissant, W Wahli, and T E Curry. 2001. \u0026ldquo;Expression and Localization of PPARs in the Rat Ovary during Follicular Development and the Periovulatory Period.\u0026rdquo; Endocrinology 142 (11) (November): 4831\u0026ndash;8. doi:10.1210/endo.142.11.8429.\u003c/p\u003e\r\n\r\n\u003cp\u003eKomar, Carolyn M, and Thomas E Curry. 2003. \u0026ldquo;Inverse Relationship between the Expression of Messenger Ribonucleic Acid for Peroxisome Proliferator-Activated Receptor Gamma and P450 Side Chain Cleavage in the Rat Ovary.\u0026rdquo; Biology of Reproduction 69 (2) (August): 549\u0026ndash;55. doi:10.1095/biolreprod.102.012831.\u003c/p\u003e\r\n\r\n\u003cp\u003eLaskey, J.W., and E. Berman. 1993. \u0026ldquo;Steroidogenic Assessment Using Ovary Culture in Cycling Rats: Effects of Bis (2-Diethylhexyl) Phthalate on Ovarian Steroid Production.\u0026rdquo; Reproductive Toxicology 7 (1) (January): 25\u0026ndash;33. doi:10.1016/0890-6238(93)90006-S.\u003c/p\u003e\r\n\r\n\u003cp\u003eLatini, Giuseppe, Egeria Scoditti, Alberto Verrotti, Claudio De Felice, and Marika Massaro. 2008. \u0026ldquo;Peroxisome Proliferator-Activated Receptors as Mediators of Phthalate-Induced Effects in the Male and Female Reproductive Tract: Epidemiological and Experimental Evidence.\u0026rdquo; PPAR Research 2008 (January): 359267. doi:10.1155/2008/359267.\u003c/p\u003e\r\n\r\n\u003cp\u003eLenie, Sandy, and Johan Smitz. 2009. \u0026ldquo;Steroidogenesis-Disrupting Compounds Can Be Effectively Studied for Major Fertility-Related Endpoints Using in Vitro Cultured Mouse Follicles.\u0026rdquo; Toxicology Letters 185 (3) (March 28): 143\u0026ndash;52. doi:10.1016/j.toxlet.2008.12.015.\u003c/p\u003e\r\n\r\n\u003cp\u003eLovekamp-Swan, Tara, and Barbara J. Davis. 2003. \u0026ldquo;Mechanisms of Phthalate Ester Toxicity in the Female Reproductive System.\u0026rdquo; Environmental Health Perspectives 111 (2) (October 28): 139\u0026ndash;145. doi:10.1289/ehp.5658.\u003c/p\u003e\r\n\r\n\u003cp\u003eLovekamp-Swan, Tara, Anton M Jetten, and Barbara J Davis. 2003. \u0026ldquo;Dual Activation of PPARalpha and PPARgamma by Mono-(2-Ethylhexyl) Phthalate in Rat Ovarian Granulosa Cells.\u0026rdquo; Molecular and Cellular Endocrinology 201 (1-2) (March 28): 133\u0026ndash;41.\u003c/p\u003e\r\n\r\n\u003cp\u003eLyche, Jan L, Arno C Gutleb, Ake Bergman, Gunnar S Eriksen, AlberTinka J Murk, Erik Ropstad, Margaret Saunders, and Janneche U Skaare. 2009. \u0026ldquo;Reproductive and Developmental Toxicity of Phthalates.\u0026rdquo; Journal of Toxicology and Environmental Health. Part B, Critical Reviews 12 (4) (April): 225\u0026ndash;49. doi:10.1080/10937400903094091.\u003c/p\u003e\r\n\r\n\u003cp\u003eMartino-Andrade, Anderson Joel, and Ibrahim Chahoud. 2010. \u0026ldquo;Reproductive Toxicity of Phthalate Esters.\u0026rdquo; Molecular Nutrition \u0026amp; Food Research 54 (1) (January): 148\u0026ndash;57. doi:10.1002/mnfr.200800312.\u003c/p\u003e\r\n\r\n\u003cp\u003eMor\u0026aacute;n, F M, A J Conley, C J Corbin, E Enan, C VandeVoort, J W Overstreet, and B L Lasley. 2000. \u0026ldquo;2,3,7,8-Tetrachlorodibenzo-P-Dioxin Decreases Estradiol Production without Altering the Enzyme Activity of Cytochrome P450 Aromatase of Human Luteinized Granulosa Cells in Vitro.\u0026rdquo; Biology of Reproduction 62 (4) (April): 1102\u0026ndash;8.\u003c/p\u003e\r\n\r\n\u003cp\u003eMor\u0026aacute;n, F M, C A VandeVoort, J W Overstreet, B L Lasley, and A J Conley. 2003. \u0026ldquo;Molecular Target of Endocrine Disruption in Human Luteinizing Granulosa Cells by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin: Inhibition of Estradiol Secretion due to Decreased 17alpha-hydroxylase/17,20-Lyase Cytochrome P450 Expression.\u0026rdquo; Endocrinology 144 (2) (March): 467\u0026ndash;73. doi:10.1210/en.2002-220813.\u003c/p\u003e\r\n\r\n\u003cp\u003eMu, Y M, T Yanase, Y Nishi, R Takayanagi, K Goto, and H Nawata. 2001. \u0026ldquo;Combined Treatment with Specific Ligands for PPARgamma:RXR Nuclear Receptor System Markedly Inhibits the Expression of Cytochrome P450arom in Human Granulosa Cancer Cells.\u0026rdquo; Molecular and Cellular Endocrinology 181 (1-2) (July 5): 239\u0026ndash;48.\u003c/p\u003e\r\n\r\n\u003cp\u003ePayne, Anita H., and Dale B. Hales. 2013. \u0026ldquo;Overview of Steroidogenic Enzymes in the Pathway from Cholesterol to Active Steroid Hormones.\u0026rdquo; Endocrine Reviews (July 1).\u003c/p\u003e\r\n\r\n\u003cp\u003ePeraza, Marjorie a, Andrew D Burdick, Holly E Marin, Frank J Gonzalez, and Jeffrey M Peters. 2006. \u0026ldquo;The Toxicology of Ligands for Peroxisome Proliferator-Activated Receptors (PPAR).\u0026rdquo; Toxicological Sciences : An Official Journal of the Society of Toxicology 90 (2) (April): 269\u0026ndash;95. doi:10.1093/toxsci/kfj062.\u003c/p\u003e\r\n\r\n\u003cp\u003eQuaedackers, M E, C E Van Den Brink, S Wissink, R H Schreurs, J A Gustafsson, P T Van Der Saag, and B B Van Der Burg. 2001. \u0026ldquo;4-Hydroxytamoxifen Trans-Represses Nuclear Factor-Kappa B Activity in Human Osteoblastic U2-OS Cells through Estrogen Receptor (ER)alpha, and Not through ER Beta.\u0026rdquo; Endocrinology 142 (3) (March): 1156\u0026ndash;66. doi:10.1210/endo.142.3.8003.\u003c/p\u003e\r\n\r\n\u003cp\u003eRak-Mardyła, Agnieszka, and Anna Karpeta. 2014. \u0026ldquo;Rosiglitazone Stimulates Peroxisome Proliferator-Activated Receptor Gamma Expression and Directly Affects in Vitro Steroidogenesis in Porcine Ovarian Follicles.\u0026rdquo; Theriogenology 82 (1) (July 1): 1\u0026ndash;9. doi:10.1016/j.theriogenology.2014.02.016.\u003c/p\u003e\r\n\r\n\u003cp\u003eSchoppee, P. D. 2002. \u0026ldquo;Putative Activation of the Peroxisome Proliferator-Activated Receptor Impairs Androgen and Enhances Progesterone Biosynthesis in Primary Cultures of Porcine Theca Cells.\u0026rdquo; Biology of Reproduction 66 (1) (January 1): 190\u0026ndash;198. doi:10.1095/biolreprod66.1.190.\u003c/p\u003e\r\n\r\n\u003cp\u003eSeto-Young, Donna, Dimiter Avtanski, Marina Strizhevsky, Grishma Parikh, Parini Patel, Julia Kaplun, Kevin Holcomb, Zev Rosenwaks, and Leonid Poretsky. 2007. \u0026ldquo;Interactions among Peroxisome Proliferator Activated Receptor-Gamma, Insulin Signaling Pathways, and Steroidogenic Acute Regulatory Protein in Human Ovarian Cells.\u0026rdquo; The Journal of Clinical Endocrinology and Metabolism 92 (6) (June): 2232\u0026ndash;9. doi:10.1210/jc.2006-1935.\u003c/p\u003e\r\n\r\n\u003cp\u003eSvechnikova, Konstantin, Irina Svechnikova, and Olle S\u0026ouml;der. 2011. \u0026ldquo;Gender-Specific Adverse Effects of Mono-Ethylhexyl Phthalate on Steroidogenesis in Immature Granulosa Cells and Rat Leydig Cell Progenitors in Vitro.\u0026rdquo; Frontiers in Endocrinology 2 (January): 9. doi:10.3389/fendo.2011.00009.\u003c/p\u003e\r\n\r\n\u003cp\u003eToda, Katsumi, Teruhiko Okada, Chisata Miyaura, and Toshiji Saibara. 2003. \u0026ldquo;Fenofibrate, a Ligand for PPARalpha, Inhibits Aromatase Cytochrome P450 Expression in the Ovary of Mouse.\u0026rdquo; Journal of Lipid Research 44 (2) (February): 265\u0026ndash;70. doi:10.1194/jlr.M200327-JLR200.\u003c/p\u003e\r\n\r\n\u003cp\u003eTreinen, K A, W C Dodson, and J J Heindel. 1990. \u0026ldquo;Inhibition of FSH-Stimulated cAMP Accumulation and Progesterone Production by mono(2-Ethylhexyl) Phthalate in Rat Granulosa Cell Cultures.\u0026rdquo; Toxicology and Applied Pharmacology 106 (2) (November): 334\u0026ndash;40.\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003e\u003cstrong\u003eBiological plausibility, coherence, and consistency of the experimental evidence\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eIn the presented AOP it is hypothesized that the key events occur in a biologically plausible order prior to the development of adverse outcomes. However, the experimental support is derived from a limited number of studies. The PPAR\u0026gamma; activators have been shown to alter steroidogenesis, ovarian cycle and impair reproduction [see reviews (Kay, Chambers, and Foster 2013), (Froment et al. 2006), (Peraza et al. 2006), (Latini et al. 2008), (Martino-Andrade and Chahoud 2010), (Lyche et al. 2009), (Lovekamp-Swan and Davis 2003)]. The biochemistry of steroidogenesis and the predominant role of the ovaries in synthesis of the sex steroids are well established. During the reproductive years the ovary is the central organ providing hormones necessary for the communication between the reproductive tract and the central nervous system, assuring normal reproductive function. Hormonal imbalance may lead to irregularities of the ovarian cycle that could be one of many possible events resulting in decrease fertility.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConcordance of dose-response relationships\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThis is a qualitative description of the pathway; the currently available studies provide little quantitative information on dose-response relationships between key events (KEs). The experimental data for selected compounds (phthalates, phenols and parabens) reveals concordance between one KE to the next in the sequence, i.e. that each KE occur at first and on lower dose than the following KE. To establish more reliable and quantitative linkages tailored experiments are required.\u003c/p\u003e\r\n\r\n\u003cp\u003eTemporal concordance among the key events and the adverse outcome\u003c/p\u003e\r\n\r\n\u003cp\u003eMost of the gathered evidence relies on the measurement of the effects at the same time point (detailed information captured in KER), thus studies providing evidence for complete temporal concordance are missing.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eStrength, consistency, and specificity of association of adverse effect and initiating event\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003ePPAR\u0026gamma;-null mutation is embryonically lethal due to a defect in placental development ( PPAR\u0026gamma; is necessary for angiogenesis)(Barak et al. 1999). Organ (ovary) targeted knock-out studies are needed to more precisely inform on the mechanistic involvement of the PPAR family in the proposed AOP.\u003c/p\u003e\r\n\r\n\u003cp\u003eThe pathway\u0026#39;s weak point lies in the linkages between the initial events in the pathway. However, there is evidence supporting both chemical dependent and independent involvement of PPAR\u0026gamma; in the female reproductive function:\u003c/p\u003e\r\n\r\n\u003cp\u003eChemical independent studies:\u003c/p\u003e\r\n\r\n\u003cp\u003e1. disruption of PPAR\u0026gamma; in ovary using cre/loxP technology led to ovarian dysfunction and female subfertility (30% of animals infertile, reminders had delayed conception and reduced litter size) (Cui et al. 2002)\u003c/p\u003e\r\n\r\n\u003cp\u003e2. granulosa cell specific deletion of PPAR\u0026gamma; in mice results in marked impairment of ovulation due to defective follicular rupture (Kim et al. 2008)\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nChemical dependent studies:\u003c/p\u003e\r\n\r\n\u003cp\u003e3. Antagonist of PPAR\u0026gamma; recovered the decrease of aromatase after treatment with MEHP (PPAR\u0026gamma; agonist) (Lovekamp-Swan, Jetten, and Davis 2003)\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nAlternative mechanism(s) or MIE(s) described which may contribute/synergise the postulated AOP\u003c/p\u003e\r\n\r\n\u003cp\u003eAlternative mechanisms relating to the pathway are described in greater detail in the descriptions of KERs.\u003c/p\u003e\r\n\r\n\u003cp\u003eThe contributing MIE in the pathway proposed is activation of PPAR\u0026alpha; supported by experimental evidence of dual activation of PPAR\u0026alpha;/\u0026gamma; by MEHP leading to decreased expression and activity of aromatase in granulosa cells (Lovekamp-Swan, Jetten, and Davis 2003) and inhibition of aromatase expression upon activation of PPAR\u0026alpha; by the ligand, fenofibrate, in the ovary of mouse (Toda et al. 2003).\u003c/p\u003e\r\n\r\n\u003cp\u003eThe relation of PPAR\u0026gamma; activation to other enzymes in steroidogenesis and reduced estradiol production PPAR\u0026gamma; ligands were shown to modulate other enzymes involved in steroidogenesis\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eupstream of aromatase:\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u0026bull; Steroidogenic acute regulatory protein (StAR)\u003c/p\u003e\r\n\r\n\u003cp\u003eStAR was up regulated by PPAR\u0026gamma; ligands (rosiglitazone and pioglitazone) in human granulosa cells in vitro (Seto-Young et al. 2007) and by MEHP in rat granulosa cells (Svechnikova, Svechnikova, and S\u0026ouml;der 2011). StAR facilitates that rapid mobilization of cholesterol for initial catalysis to pregnenolone by the P450-side chain cleavage enzyme located within the mitochondria ( see review (Payne and Hales 2013)).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026bull; 3\u0026beta;-hydroxysteroid dehydrogenase (3\u0026beta;-HSD)\u003c/p\u003e\r\n\r\n\u003cp\u003eContradictory results were found on the effect of PPAR\u0026gamma; ligands on 3\u0026beta;-HSD enzyme. Work on porcine granulosa cells has found that troglitazone competitively inhibits 3\u0026beta;-HSD enzyme activity (Gasic et al. 1998). Opposite results were obtained with another agonist of PPAR\u0026gamma; (rosiglitazone) that stimulated 3\u0026beta;HSD protein expression and activity in porcine ovarian follicles (Rak-Mardyła and Karpeta 2014). 3\u0026beta;-HSD catalyses the conversion of pregnenolone to progesterone see review (Payne and Hales 2013)\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026bull; 17-alpha-hydroxylase (P450c17, CYP 17) Conflicting reports have arisen regarding the effect of PPAR\u0026gamma; agonists on the expression and activity of this enzyme, mRNA production was unchanged following porcine thecal cell exposure to PPAR\u0026gamma; ligand (Schoppee 2002), whilst other reports indicate CYP17 expression inhibition by PPAR\u0026gamma; (rosiglitazone) agonist in ovarian follicles (Rak-Mardyła and Karpeta 2014). P450c17converts progesterone to androgen see review (Payne and Hales 2013)\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003edownstream of aromatase:\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003eReduced production of estradiol may result from alteration of the enzymes upstream of aromatase (described above) or by increasing estradiol catabolism (altering Cyp1b1 and 17-\u0026beta;HSD IV, which are involved in estradiol conversion to catechol estrogens and estrone respectively).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n\u0026bull; 17\u0026beta;-Hydroxysteroid dehydrogenase (17\u0026beta;-HSD)\u003c/p\u003e\r\n\r\n\u003cp\u003eAgonist of PPAR\u0026gamma; (rosiglitazone) was found to inhibit 17\u0026beta;-HSD protein expression in ovarian follicles (Rak-Mardyła and Karpeta 2014), whereas increase in enzyme expression was noted upon treatment of granulosa cells by phthalate (MEHP) (Lovekamp-Swan, Jetten, and Davis 2003). 17\u0026beta;-Hydroxysteroid dehydrogenase (17\u0026beta;-HSD) metabolises estradiol to estrone see review (Payne and Hales 2013). For example, in vitro studies with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) investigating steroid production in human luteinizing granulosa cells (hLGC) showed estradiol decreased without changing either aromatase protein or its enzyme activity (Mor\u0026aacute;n et al. 2000). Studies by the same laboratory identified P450c17 as a molecular target for endocrine disruption of hLGC specifically decreasing the supply of androgens for E2 synthesis (Mor\u0026aacute;n et al. 2003). Reduced levels of estradiol production may result from increased inactivation of E2 via conversion to estrone as shown in isolated mouse small preantral follicles upon phthalate (MEHP) treatment (Lenie and Smitz 2009) and granulosa cells (Lovekamp-Swan, Jetten, and Davis 2003). Taken together, these findings provide strong evidence for the direct effect of PPAR\u0026gamma; agonists on ovarian synthesis and secretion of hormones.\u003c/p\u003e\r\n\r\n\u003cp\u003eReduced levels of estradiol and irregularities of ovarian cycle\u003c/p\u003e\r\n\r\n\u003cp\u003eThe impact on ovarian cycle may result from a defect in hypothalamic-pituitary-gonadal (HPG) axis signalling, other than by alteration of estradiol level. MEHP inhibited follicle-simulating hormone (FSH) mediated stimulation of adenylate cyclase and progesterone synthesis in primary cultures of rat granulosa cells (Treinen, Dodson, and Heindel 1990).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n\u003cstrong\u003eUncertainties, inconsistencies and data gaps\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThe current major uncertainty in this AOP is the basis of the functional relationship between the PPAR\u0026gamma;, activation leading to Aromatase (Cyp19a1), reduction in ovarian granulosa cells. The possible mechanisms have been proposed and investigated, however there is lack of dose response and temporal data supporting the relationship (Lovekamp-Swan, Jetten, and Davis 2003), (Fan et al. 2005), (Mu et al. 2001). The pattern of the PPAR\u0026gamma; expression in ovarian follicles is not steady, unlike expression of PPAR\u0026alpha; and \u0026delta;. This fact adds to the complexity to the interpretation of mechanisms involved in the pathway. The PPAR\u0026gamma; is down-regulated in response to the LH surge (C M Komar et al. 2001), but only in follicles that have responded to the LH surge (Carolyn M Komar and Curry 2003). Because PPAR\u0026gamma; is primarily expressed in granulosa cells, it may influence development of these cells and their ability to support normal oocyte maturation. PPAR\u0026gamma; could also potentially affect somatic cell/oocyte communication not only by impacting granulosa cell development, but by direct effects on the oocyte. Modulation of the PPAR\u0026gamma; activity/expression in the ovary therefore, could potentially affect oocyte developmental competence. There is high strength, as well as specificity starting from the association between the reductions of E2 production leading to fertility impairment in females. Consistency of key events in the AOP is supported by several lines of evidence deriving from in vitro and in vivo studies that support PPAR\u0026gamma; activation as an important actor in reproductive toxicity in rodents [(Kay, Chambers, and Foster 2013), (Froment et al. 2006), (Peraza et al. 2006), (Latini et al. 2008), (Martino-Andrade and Chahoud 2010), (Lyche et al. 2009), (Lovekamp-Swan and Davis 2003)].\u003c/p\u003e\r\n\r\n\u003cp\u003eInconsistencies\u003c/p\u003e\r\n\r\n\u003cp\u003eAgonists of PPAR\u0026gamma; were found to impact on steroidogenesis; however contradictory data show their effect on different stages of the process as well the direction of the effect(see above). Some in vivo studies also reported two-way effect on the estradiol production by PPAR\u0026gamma; agonists. This effect may be attributed to the different measurements during different stages of estrous cycle. The phase of the estrous cycle, in which hormones are measured, may influence the readout of compound effect. In rats treated with DEHP increase in estradiol production was observed in ovarian cells (ex vivo) extracted during diestrus phase, however there was decrease in estradiol when the cells were extracted during estrus stage (Laskey and Berman 1993). In alignment with this result increased levels of estradiol were found in sheep proceeding the estrus phase (Herreros et al. 2013).\u003c/p\u003e\r\n\r\n\u003cp\u003eData Gaps: There is a limited number of studies investigating the effect of PPAR\u0026gamma; and its role in female reproductive function, in order to establish a more quantitative and temporal coherent linkage of the MIE to the subsequent key events studies are required. For example: the plausible mechanism of activation of a PPAR\u0026gamma;, RXR and involvement of NFkappaB and their role in transcriptional repression of the aromatase gene could be investigated in modified transactivation assays to measure NFkappaB repression, rather than transactivation. Similar assays have been already generated, for estrogen receptor-mediated transrepression (Quaedackers et al. 2001).\u003c/p\u003e\r\n","background":"","user_defined_mie":"408: reduction in ovarian granulosa cells, Aromatase (Cyp19a1)","user_defined_ao":"406: impaired, Fertility and 405: irregularities, ovarian cycle","oecd_project":"1.21","oecd_status_id":3,"graphical_representation_image_uid":"2016/11/29/009273188_PPAR_activation_leading_to_reproductive_toxicity.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":null,"development_strategy":null,"known_modulating_factors":null,"assigned_license_id":6,"handbook_id":1,"project_129":false},{"id":8,"title":"Upregulation of Thyroid Hormone Catabolism via Activation of Hepatic Nuclear Receptors, and Subsequent Adverse Neurodevelopmental Outcomes in Mammals","short_name":"Nuclear receptor induced TH Catabolism and Developmental Hearing Loss","corresponding_author_id":181,"abstract":"\u003cp\u003eData from rodent studies demonstrate that thyroid hormone disruption during cochlear development culminates in ototoxicity. Developmental exposure of rats to polychlorinated biphenyls (PCBs) results in a low-frequency hearing loss in adult offspring (Goldey et al., 1995a; Herr et al., 1996; 2001; Crofton and Rice, 1999; Laskey et al., 2002). A body of work now supports the hypothesis that this ototoxicity results from PCB-induced hypothyroxinemia during a critical period of auditory development. Evidence for this hypothesis includes: a correlation between the severity of functional auditory impairment and the degree of thyroid hormone depletion (Goldey et al., 1995a; 1995b; Goldey and Crofton, 1998; Crofton, 2004), a cross-fostering study demonstrating that the critical exposure period is postnatal (Crofton et al., 2000a), and amelioration of the hearing loss following postnatal thyroxine replacement (Goldey and Crofton, 1998). Below an adverse outcome pathway is described for chemicals that activate xenobiotic nuclear receptors, including AhR, CAR, and PXR, leading to thyroid hormone disruption during cochlear development and resulting in permanent auditory loss.\u003c/p\u003e\r\n\r\n\u003cp\u003eThis AOP is a revision and update of the original started on the Chemical Mode of Action wiki sponsored by WHO/IPCS. This MOA was described and published by Crofton and Zoeller 2005).\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":3,"authors":"\u003cp\u003eKatie Paul Friedman, National Center for Computational Toxicology, US EPA, RTP, NC USA \u0026lt;paul-frieidman@epa.gov\u0026gt;\u003c/p\u003e\r\n\r\n\u003cp\u003eMary E. Gilbert, National Health and Environmental Effects Research Laboratory, US EPA, RTP, NC USA \u0026lt;gilbert.mary@epa.gov\u0026gt;\u003c/p\u003e\r\n\r\n\u003cp\u003eKevin M. Crofton, National Center for Computational Toxicology, US EPA, RTP, NC USA \u0026lt;crofton.kevin@epa.gov\u0026gt;\u003c/p\u003e\r\n","applicability_of_the_aop":"","key_event_essentiality":"","weight_of_evidence_summary":"\u003cp\u003e\u003ca href=\"#Relationships_Among_Key_Events_and_the_Adverse_Outcome\"\u003eSummary Table\u003c/a\u003e\u003cbr /\u003e\r\n\u003cem\u003eProvide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages. \u003c/em\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConcordance of dose-response relationships\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eMultiple studies provide limited (2-3 doses) dose-response data for many of the key events. These studies demonstrate similar magnitudes of effect on circulating hormones for doses of PCBs that are within an order of magnitude (3-25 mg/kg/day for Aroclor 1254) (e.g.,Morse et al., 1996; Goldey et al., 1998). Very limited data are available correlating any of the key events. One exception is the relationship between circulating serum T4 concentrations during development and the magnitude of hearing loss (Crofton, 2004). There is a very good correlation between total serum T4 concentrations on postnatal day (PND) 14 and hearing loss assessed in adult offspring of PCB exposed dams (Figure 2). All of these events occur within a 2-3 fold dose range.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nTemporal concordance among the key events and the adverse outcome Strength, consistency, and specificity of association of adverse effect and initiating event Biological plausibility, coherence, and consistency of the experimental evidence Alternative mechanism(s) or MIE(s) that logically present themselves and the extent to which they may detract from the AOP Uncertainties, inconsistencies, and data gaps\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","quantitative_considerations":"","optional_considerations":"","references":"","overall_assessment":"","background":"\u003cp\u003eThis AOP is an update of the WHO/IPCS MOA developed in 2005 by Crofton and Zoeller (Crit Rev Toxicol 2005).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cem\u003eCrofton KM, Zoeller RT. Mode of action: neurotoxicity induced by thyroid hormone disruption during development--hearing loss resulting from exposure to PHAHs. Crit Rev Toxicol. 2005 Oct-Nov;35(8-9):757-69. PMID: 6417043\u003c/em\u003e\u003c/p\u003e\r\n","user_defined_mie":"239: Activation, Pregnane-X receptor, NR1l2","user_defined_ao":"319: Loss, Cochlear function","oecd_project":"1.9","oecd_status_id":4,"graphical_representation_image_uid":"2016/11/29/448305262_Nuclear_receptor_activation_and_ototoxicity.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2021-09-14T12:35:30.000-04:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":7,"handbook_id":1,"project_129":false},{"id":10,"title":"Binding to the picrotoxin site of ionotropic GABA receptors leading to epileptic seizures in adult brain","short_name":"Blocking iGABA receptor ion channel leading to seizures","corresponding_author_id":261,"abstract":"\u003cp\u003eThis AOP begins with a molecular initiating event (MIE) where a chemical binds to the picrotoxin binding site at or near the central pore of the ionotropic GABA receptor complex causing blockage of the ion channel. As a result, the first key event (KE)\u0026nbsp;is a decrease in inward chloride conductance through the ligand-gated ion channel. This leads to the second KE, a reduction in postsynaptic inhibition, reflected as reduced frequency and amplitude of spontaneous inhibitory postsynaptic current (sIPSC) or abolishment of GABA-induced firing action in GABAergic neuronal membranes. Consequently, the resistance of excitatory neurons to fire is decreased, resulting in the generation of a large excitatory postsynaptic potential (EPSP), i.e., the third KE.\u0026nbsp;The large EPSP is reflected as a\u0026nbsp;spike (rise) of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e observed in the affected region, where a large group of excitatory neurons begin firing in an abnormal, excessive, and synchronized manner. Such a giant Ca\u003csup\u003e2+\u003c/sup\u003e-mediated excitatory firing (depolarization) causes voltage-gated Na\u003csup\u003e+\u003c/sup\u003e to open, which results in action potentials. The depolarization is followed by a period of hyper-polarization mediated by Ca\u003csup\u003e2+\u003c/sup\u003e-dependent K\u003csup\u003e+\u003c/sup\u003e channels or GABA-activated Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e influx. During seizure development, the post-depolarization hyperpolarization becomes smaller, gradually disappears, and is replaced by a depolarization. This characteristic depolarization-shrinking hyperpolarization sequence\u0026nbsp;of events represents the fourth KE known as \u0026ldquo;paroxysmal depolarizing shift\u0026rdquo; (PDS), which forms a \u0026ldquo;seizure focus\u0026rdquo;. A PDS is, essentially, an indication of epilepsy at the cellular level, which serves as the foci to initiate the adverse outcome at the organismal level of epileptic seizure. The severity of symptoms is often dose- and duration- dependent, while the toxicological symptoms are associated with the type and location of affected iGABARs. Mortality can occur if the individual sustains a prolonged or pronounced convulsion or seizure. Neurotoxicity, of which seizures\u0026nbsp;are\u0026nbsp;an end point, is a regulated outcome for chemicals. This AOP allows for screening chemicals for the potential to cause neurotoxicity through the use of \u003cem\u003ein vitro \u003c/em\u003eassays that demonstrate binding to the picrotoxin site, electrophysiological assays demonstrating depolarization of neuronal membranes, or electroencephalography that records electrical activity of the adult brain.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":1,"authors":"\u003cp\u003ePing Gong, Edward J. Perkins, US Army Engineer Research and Development Center\u003c/p\u003e\r\n\r\n\u003cp\u003eEmail: ping.gong@usace.army.mil or edward.j.perkins@usace.army.mil\u003c/p\u003e\r\n\r\n\u003cp\u003ePoint of contact for this AOP entry: Dr. Ping Gong\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003eThis AOP is applicable to all vertebrates and invertebrates possessing iGABARs, without restrictions pertaining to sex and taxonomy. This AOP may not be applicable to young animals during their embryonic and early developmental stages\u0026nbsp;when GABA acts as an excitatory neurotransmitter due to increased intracellular Cl\u003csup\u003eˉ\u003c/sup\u003e concentration in immature\u0026nbsp;or developing neurons\u0026nbsp;(Taketo and Yoshioka 2000; Galanopoulou 2008; Ben-Ari 2006). A key feature of the immature type function of GABA\u003csub\u003eA\u003c/sub\u003e receptors is the depolarizing signaling, attributed to the inability of young neurons to maintain low intracellular chloride. The regulation of GABAergic switch is different in neurons with depolarizing vs hyperpolarizing GABAergic signaling. In mature neurons, recurrent and prolonged seizures may trigger a pathological reemergence of immature features of GABA\u003csub\u003eA\u003c/sub\u003e receptors, which compromises the efficacy of GABA-mediated inhibition. In immature neurons with depolarizing GABAergic signaling, the physiological and pathological regulation of this system is completely different, possibly contributing to the different outcomes of early life seizures (Galanopoulou 2008).\u003c/p\u003e\r\n","key_event_essentiality":"\u003cp\u003eThe MIE, four key events and resulted adverse outcome listed for this AOP are all essential based on current knowledge and understanding of the structure, pharmacology, localization, classification of ionotropic GABA receptors (e.g., GABA\u003csub\u003eA\u003c/sub\u003e receptors) (Olsen 2015; Olsen and Sieghart 2009), the basic neurophysiology, neurochemistry and cellular mechanisms underlying epilepsies (Dichter and Ayala 1987; Bromfield \u003cem\u003eet al\u003c/em\u003e. 2006), and the pathophysiology of seizures (Lomen-Hoerth and Messing 2010).\u003c/p\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003eA novel subject matter expertise driven approach was developed for weight of evidence (WoE) assessment (Collier \u003cem\u003eet al\u003c/em\u003e. 2016). This approach, tailored toward the needs of AOPs and in compliance with the AOP Users\u0026#39; Handbook (OECD 2017), was based on criteria and metrics related to data quality and causality (i.e., the strength of causal linkage between key events). The methodology consists of three main steps: (1) assembling evidence (preparing the AOP), (2) weighting evidence (criteria weighting and scoring), and (3) weighting the body of evidence (aggregating lines of evidence). We adopted the General Assessment Factors (GAF) established by the US EPA as the criteria for data quality evaluation, and a set of five criteria known as Bradford Hill criteria to measure the strength of causal linkages (see Table below). The numerical scoring\u0026nbsp;scale corresponds to the descriptive scoring scale recommended in the Users\u0026#39; Handbook as follows: 4 or 5 is equivalent to High, 3 is equivalent to Moderate, and 1 or 2 is equivalent to Low.\u0026nbsp;Detailed description on, supporting evidence for and quantitative understanding of\u0026nbsp;each KE/KER can be found by clicking the link on each KE/KER above.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eThe authors of Collier \u003cem\u003eet al\u003c/em\u003e. (2016), who served as the developers for several AOPs (including this one), represented subject matter experts, and they applied their expertise and best professional judgment to assign weights to the criteria and scores to each line of evidence. Final criteria scoring represented the consensus scores agreed upon after debates among the authors. For example, the MIE has been intensively reviewed where numerous documented studies provided supporting evidence. Hence, the MIE received high scores for all five GAF criteria. However, the Bradford Hill criteria connecting KE2--\u0026gt;KE3 and KE3--\u0026gt;KE4 received relatively lower scores because there still exist knowledge gaps in the spread of epileptic activity throughout the normal CNS and the mechanism underlying the generalized epilepsies. The following table shows the results of our WoE assessment (note that scores may be inexact due to rounding).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003ca class=\"image\" href=\"/wiki/index.php/File:ScoreTable.jpg\"\u003e\u003cimg alt=\"ScoreTable.jpg\" src=\"/wiki/images/c/cd/ScoreTable.jpg\" style=\"height:701px; width:1350px\" /\u003e\u003c/a\u003e\u003c/p\u003e\r\n","quantitative_considerations":"\u003cp\u003eMany studies have reported quantitative relationships between chemicals such as drugs and pesticides and electrophysiological response. For instance, long-term exposure of primary cerebellar granule cell cultures to 3 \u0026micro;M dieldrin reduced the GABA\u003csub\u003eA\u003c/sub\u003e receptor function to 55% of control, as measured by the GABA-induced \u003csup\u003e36\u003c/sup\u003eCl\u003csup\u003e-\u003c/sup\u003e uptake (Babot \u003cem\u003eet al\u003c/em\u003e. 2007). Juarez \u003cem\u003eet al\u003c/em\u003e. (2013) observed that picrotoxin exerted concentration-dependent and reversible inhibition of GABA-induced membrane currents in primary cultured neurons obtained from the guinea-pig small intestine. The stepwise qualitative relationships between consecutive events (MIE, KEs and AO) are well established but quantitative ones are rarely documented.\u003c/p\u003e\r\n","optional_considerations":"\u003cp\u003eThis AOP can be used to establish the mode of neurotoxicological actions for chemicals capable of binding to the picrotoxin convulsant site of iGABARs. It can also be applied to risk assessment where AOP can assist in predictive modeling of chemical toxicity. Chemicals acting through this AOP can be distinguished from neurotoxicants acting on other types of iGABAR sites (e.g., orthosteric or allosteric binding sites) or other types of neuroreceptors (e.g., ardrenergic, dopaminergic, glutaminergic, cholinergic and serotonergic receptors). More information relevant to this topic can be found in Gong \u003cem\u003eet al\u003c/em\u003e. (2015).\u003c/p\u003e\r\n","references":"\u003cp\u003eBabot Z, Vilaro M T, Sunol C. (2007) Long-term exposure to dieldrin reduces gamma-aminobutyric acid type A and N-methyl-D-aspartate receptor function in primary cultures of mouse cerebellar granule cells. J Neurosci Res, 85(16):3687-3695.\u003c/p\u003e\r\n\r\n\u003cp\u003eBen-Ari Y. (2006) Seizures beget seizures: the quest for GABA as a key player. Crit Rev Neurobiol. 18(1-2):135-44.\u003c/p\u003e\r\n\r\n\u003cp\u003eBromfield EB, Cavazos JE, Sirven JI. (2006) Chapter 1, Basic Mechanisms Underlying Seizures and Epilepsy. In: An Introduction to Epilepsy [Internet]. West Hartford (CT): American Epilepsy Society; Available from: \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/books/NBK2510/\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/books/NBK2510/\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eCasida JE, Durkin KA. (2015) Novel GABA receptor pesticide targets.\u0026nbsp;Pesticide Biochem Physiol. 121:22-30.\u003c/p\u003e\r\n\r\n\u003cp\u003eChen L, Durkin KA, Casida J. (2006) Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site. Proc Natl Acad Sci USA.\u0026nbsp;103(13):5185-5190.\u003c/p\u003e\r\n\r\n\u003cp\u003eCole\u0026nbsp;LM, Casida JE. (1986) Polychlorocycloalkane insecticide-induced convulsions in mice in relation to disruption of the GABA-regulated chloride ionophore. Life Sci.\u0026nbsp;39 (20):1855-62.\u003c/p\u003e\r\n\r\n\u003cp\u003eCollier ZA, Gust KA, Gonzalez-Morales B, Gong P, Wilbanks MS, Linkov I, Perkins EJ. (2016) A weight of evidence assessment approach for adverse outcome pathways. Regul\u0026nbsp;Toxicol\u0026nbsp;Pharmacol.\u0026nbsp;75:46-57.\u003c/p\u003e\r\n\r\n\u003cp\u003eDichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: A status report. Science 237: 157-164.\u003c/p\u003e\r\n\r\n\u003cp\u003eDurukan P, Ozdemir C, Coskun R, Ikizceli I, Esmaoglu A, Kurtoglu S, Guven M. (2009) Experiences with endosulfan mass poisoning in rural areas. Eur J Emerg Med. 16(1):53-6.\u003c/p\u003e\r\n\r\n\u003cp\u003eGalanopoulou AS. (2008) GABA\u003csub\u003eA\u003c/sub\u003e\u0026nbsp;Receptors in Normal Development and Seizures: Friends or Foes? Curr Neuropharmacol. 6(1): 1\u0026ndash;20.\u003c/p\u003e\r\n\r\n\u003cp\u003eGarcia-Reyero N, Habib T, Pirooznia M, Gust KA, Gong P, Warner C, Wilbanks M, Perkins E. (2011) Conserved toxic responses across divergent phylogenetic lineages: a meta-analysis of the neurotoxic effects of RDX among multiple species using toxicogenomics. Ecotoxicology. 20(3):580-94.\u003c/p\u003e\r\n\r\n\u003cp\u003eGong P, Hong H, Perkins EJ. (2015) Ionotropic GABA receptor antagonism-induced adverse outcome pathways for potential neurotoxicity biomarkers. Biomarkers in Medicine 9(11):1225-39.\u003c/p\u003e\r\n\r\n\u003cp\u003eHan HA, Cortez MA, Snead OC III. 2012. GABA\u003csub\u003eB\u003c/sub\u003e Receptor and Absence Epilepsy. In: Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper\u0026#39;s Basic Mechanisms of the Epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology Information (US); 2012.\u0026nbsp;Available from: https://www.ncbi.nlm.nih.gov/books/NBK98192/.\u003c/p\u003e\r\n\r\n\u003cp\u003eHosie AM, Aronstein K, Sattelle DB, ffrench-Constant RH.\u0026nbsp;(1997) Molecular biology of insect neuronal GABA receptors. Trends Neurosci. 20(12): 578-583.\u003c/p\u003e\r\n\r\n\u003cp\u003eIkeda T, Nagata K, Shono T, Narahashi T. (1998) Dieldrin and picrotoxinin modulation of GABA(A) receptor single channels. Neuroreport 9(14):3189-3195.\u003c/p\u003e\r\n\r\n\u003cp\u003eJuarez EH, Ochoa-Cortes F, Miranda-Morales M, Espinosa-Luna R, Montano L M, Barajas-Lopez C. (2013) Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons. Auton Autacoid Pharmacol, 34(1-2):1-8.\u003c/p\u003e\r\n\r\n\u003cp\u003eLomen-Hoerth C, Messing RO. (2010) Chapter 7: Nervous system disorders. In: Stephen J. McPhee, and Gary D. Hammer (Eds), Pathophysiology of disease: an introduction to clinical medicine (6th Edition). New York: McGraw-Hill Medical. \u003ca class=\"internal mw-magiclink-isbn\" href=\"/wiki/index.php/Special:BookSources/9780071621670\"\u003eISBN 9780071621670\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eMcGonigle I, Lummis SC. (2010) Molecular characterization of agonists that bind to an insect GABA receptor. Biochemistry. 49(13):2897-902.\u003c/p\u003e\r\n\r\n\u003cp\u003eMoon JM, Chun BJ, Lee SD. (2017) In-hospital outcomes and delayed neurologic sequelae of seizure-related endosulfan poisoning. Seizure. 51:43-49.\u003c/p\u003e\r\n\r\n\u003cp\u003eMoses V, Peter JV. (2010)\u0026nbsp;Acute intentional toxicity: endosulfan and other organochlorines. Clin Toxicol (Phila). 48(6):539-44.\u003c/p\u003e\r\n\r\n\u003cp\u003eNewland CF, Cull-Candy SG. (1992) On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. J Physiol, 447: 191\u0026ndash;213.\u003c/p\u003e\r\n\r\n\u003cp\u003eOECD. (2017)\u0026nbsp;Users\u0026#39; Handbook Supplement to the Guidance Document for Developing and Assessing AOPs.\u0026nbsp;OECD Environment Directorate, Environment, Health and Safety Division,\u0026nbsp;Series on Testing \u0026amp; Assessment No. 233, Series on Adverse Outcome Pathways No. 1,\u0026nbsp;ENV/JM/MONO(2016)12, Paris, France.\u003c/p\u003e\r\n\r\n\u003cp\u003eOlsen RW. (2015) Allosteric ligands and their binding sites define \u0026gamma;-aminobutyric acid (GABA) type A receptor subtypes. Adv Pharmacol. 73:167-202.\u003c/p\u003e\r\n\r\n\u003cp\u003eOlsen RW, Sieghart W. (2009) GABA A receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology. 56(1):141-8.\u003c/p\u003e\r\n\r\n\u003cp\u003eParbhu B, Rodgers G, Sullivan JE. (2009) Death in a toddler following endosulfan ingestion. Clin Toxicol (Phila). 47(9):899-901.\u003c/p\u003e\r\n\r\n\u003cp\u003eRaymond-Delpech V, Matsuda K, Sattelle BM, Rauh JJ, Sattelle DB. (2005) Ion channels: molecular targets of neuroactive insecticides. Invert Neurosci, 5(3-4):119-133.\u003c/p\u003e\r\n\r\n\u003cp\u003eRoberts DM, Dissanayake W, Sheriff MHR, Eddleston M. (2004) Refractory status epilepticus following self-poisoning with the organochlorine pesticide endosulfan. J Clinical Neurosci. 11(7): 760-2.\u003c/p\u003e\r\n\r\n\u003cp\u003eScharfman HE, Brooks-Kayal AR. (2014) Is Plasticity of GABAergic Mechanisms Relevant to Epileptogenesis? In: Scharfman HE and Buckmaster PS (eds.), Issues in Clinical Epileptology: A View from the Bench, Advances in Experimental Medicine and Biology 813, pp.133-150.\u003c/p\u003e\r\n\r\n\u003cp\u003eSieghart W.(1995) Structure and pharmacology of gamma-aminobutyric acid A receptor subtypes. Pharmacol.Rev. 47(2):181-234\u003c/p\u003e\r\n\r\n\u003cp\u003eStilwell GE, Saraswati S, Littleton JT, Chouinard SW. (2006) Development of a Drosophila seizure model for in vivo high-throughput drug screening. Eur J Neurosci, 24(8):2211-22.\u003c/p\u003e\r\n\r\n\u003cp\u003eTaketo M, Yoshioka T (2000) Developmental change of GABA(A) receptor-mediated current in rat hippocampus. Neuroscience 96(3):507-514.\u003c/p\u003e\r\n\r\n\u003cp\u003eTreiman DM. (2001) GABAergic mechanisms in epilepsy. Epilepsia, 42(Suppl. 3):8\u0026ndash;12.\u003c/p\u003e\r\n\r\n\u003cp\u003eWilliams LR, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et al. (2011) RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures. Environ Health Perspect, 119(3):357-363.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003e\u003cstrong\u003eBiological plausibility\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThe biological mechanisms underlying epilepsy (defined as a disorder of the central nervous system characterized by recurrent seizures unprovoked by an acute systemic or neurologic insult) have been investigated for more than six decades and are well understood except for a few intermediate details (Bromfield \u003cem\u003eet al\u003c/em\u003e. 2006; Lomen-Hoerth and Messing 2010). As one of the cellular mechanisms of action, blocking postsynaptic GABA-mediated inhibition can lead to epileptic seizure (Dichter and Ayala 1987; Gong \u003cem\u003eet al\u003c/em\u003e. 2015). It has been extensively documented that non-competitive ion channel blockers such as picrotoxin, lindane, \u0026alpha;-endosulfan and fipronil act through binding to iGABARs (Chen \u003cem\u003eet al\u003c/em\u003e. 2006; Casida and Durkin 2015). Despite large structural diversity, it has been postulated that these blockers fit a single binding site in the chloride channel lumen lined by five TM2 (TransMembrane domain 2) segments, which was supported in the \u0026beta;3 homopentamer by mutagenesis, pore structure studies, ligand binding, and molecular modeling (Chen \u003cem\u003eet al\u003c/em\u003e. 2006; Casida and Durkin 2015). The downstream cascading key events of this AOP have also been reviewed in multiple publications (e.g., Dichter and Ayala 1987; Bromfield \u003cem\u003eet al\u003c/em\u003e. 2006; Lomen-Hoerth and Messing 2010). Based on the extensive evidence supporting the MIE, KEs and the AO, there is a high likelihood and certainty that such GABA antagonists as non-competitive channel blockers produce seizures in both invertebrates and vertebrates that possess GABAergic inhibitory neurotransmission in central nervous systems (Treiman 2001; Raymond-Delpech \u003cem\u003eet al\u003c/em\u003e. 2005).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConcordance of dose-response relationships\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eNumerous pharmacological studies have reported quantitative dose-response relationships between the dose of non-competitive antagonists and the recorded electrophysiological response of epileptic seizures. See examples for picrotoxin (Newland and Cull-Candy 1992; Ikeda 1998; Stilwell \u003cem\u003eet al\u003c/em\u003e. 2006), RDX (Williams \u003cem\u003eet al\u003c/em\u003e. 2011) and dieldrin (Babot \u003cem\u003eet al\u003c/em\u003e. 2007; Ikeda 1998).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eTemporal concordance among the key events and the adverse outcome\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eGiven that the basic mechanism of neuronal excitability is the action potential, a hyperexcitable state can result from many causes including decreased inhibitory neurotransmission (KE2). Moreover, action potentials occur due to depolarization of the neuronal membrane, with membrane depolarization propagating down the axon to induce neurotransmitter release at the axon terminal. The action potential occurs in an all-or-none fashion as a result of local changes in membrane potential brought about by net positive inward ion fluxes. Membrane potential thus varies with the activation of ligand-gated channels, whose conductance is affected by binding to neurotransmitters. For instance, the conductance is decreased (KE1) due to the binding at allosteric sites in the chloride channel of iGABAR by non-competitive blockers (MIE).\u003c/p\u003e\r\n\r\n\u003cp\u003eSeizure initiation: The hypersynchronous discharges that occur during a seizure may begin in a very discrete region of the cortex and then spread to neighboring regions. Seizure initiation is characterized by two concurrent events: 1) high-frequency bursts of action potentials, and 2) hypersynchronization of a neuronal population. The synchronized bursts from a sufficient number of neurons result in a so-called spike discharge on the EEG (electroencephalogram), i.e., amplified excitatory postsynaptic potential (KE3). At the level of single neurons, epileptiform activity consists of sustained neuronal depolarization resulting in a burst of action potentials, a plateau-like depolarization associated with completion of the action potential burst, and then a rapid repolarization followed by hyperpolarization. This sequence is called the paroxysmal depolarizing shift (KE4).\u003c/p\u003e\r\n\r\n\u003cp\u003eSeizure propagation (AO), the process by which a partial seizure spreads within the brain, occurs when there is sufficient activation to recruit surrounding neurons. This leads to a loss of surround inhibition and spread of seizure activity into contiguous areas via local cortical connections, and to more distant areas via long association pathways such as the corpus callosum. The propagation of bursting activity is normally prevented by intact hyperpolarization and a region of surrounding inhibition created by inhibitory neurons. With sufficient activation there is a recruitment of surrounding neurons via a number of mechanisms. The above description is excerpted and summarized from Bromfield \u003cem\u003eet al\u003c/em\u003e. (2006).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eStrength, consistency, and specificity of association of adverse effect and initiating event\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eDrug- or chemical-induced focal or generalized seizures are not limited to any specific group of chemical structures, neuroreceptors or taxonomy. This AOP addresses a specific group of chemicals that are capable of binding to the picrotoxin convulsant site of iGABARs, leading to epileptic seizures. Literature evidence strongly and consistently supports such a forward association, i.e., binding to the picrotoxin site leads to epileptic seizures (see reviews Gong \u003cem\u003eet al\u003c/em\u003e. 2015; Bromfield \u003cem\u003eet al\u003c/em\u003e. 2006; Raymond-Delpech \u003cem\u003eet al\u003c/em\u003e. 2005; Treiman 2001; Dichter and Ayala 1987). For instance,\u0026nbsp;dose- and time-dependent correlations between picrotoxin site binding by\u0026nbsp;chlorinated pesticides and convulsions were observed in mice (Cole and Casida 1986), whereas poisoning\u0026nbsp;with the organochlorine insecticide endosulfan caused\u0026nbsp;seizure, status epilepticus, or refractory\u0026nbsp;status epilepticus in humans (Durukan \u003cem\u003eet al\u003c/em\u003e.\u0026nbsp;2009; Moon \u003cem\u003eet\u0026nbsp;al\u003c/em\u003e.\u0026nbsp;2017; Moses and Peter\u0026nbsp;2010; Parbhu \u003cem\u003eet al\u003c/em\u003e.\u0026nbsp;2009; Roberts \u003cem\u003eet al\u003c/em\u003e. 2004),\u0026nbsp;and eventually led to the death of a farmer (Roberts \u003cem\u003eet al\u003c/em\u003e. 2004) and a toddler (Parbhu \u003cem\u003eet al\u003c/em\u003e. 2009).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eUncertainties, inconsistencies, and data gaps \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eNo inconsistencies have been reported so far, though some uncertainties and data gaps do exist. For instance, the process by which seizures typically end, usually after seconds or minutes, and what underlies the failure of this spontaneous seizure termination in the life-threatening condition known as status epilepticus are\u0026nbsp;less well understood\u0026nbsp;(Bromfield \u003cem\u003eet al\u003c/em\u003e. 2006). The spread of epileptic activity throughout the brain, the development of primary generalized epilepsy, the existence of \u0026ldquo;gating\u0026quot;,\u0026nbsp;mechanisms in specific anatomic locations, and the extrapolation of hypotheses derived from simple models of focal epilepsy to explain more complex forms of epilepsies observed in human and other animals, all are also not yet fully understood (Dichter and Ayala 1987).\u0026nbsp;The remarkable plasticity of GABAergic neurons and iGABARs (e.g., iGABAR regulation by phosphorylation, and expression of potassium chloride cotransporters (KCCs) or sodium dependent anion exchangers (NDAE)) in response to insults and injury constitutes additional complexity and\u0026nbsp;creates\u0026nbsp;another layer of uncertainties\u0026nbsp;in the emergence of epileptic seizures\u0026nbsp;(Ben-Ari 2006; Galanopoulou 2008; Scharfman and Brooks-Kayal 2014).\u003c/p\u003e\r\n","background":"\u003cp\u003eIonotropic GABA receptors (iGABARs) are ligand-gated ion channels which play important functional roles in the nervous system. As the major player in inhibitory neurotransmission, iGABARs are widely distributed in\u0026nbsp;both vertebrates and invertebrates (McGonigle\u0026nbsp;and Lummis 2010; Garcia-Reyero \u003cem\u003eet al\u003c/em\u003e. 2011). In vertebrates, the iGABAR includes two subclasses of fast-responding ion channels, GABA\u003csub\u003eA\u003c/sub\u003e receptor (GABA\u003csub\u003eA\u003c/sub\u003e-R) and GABA\u003csub\u003eC\u003c/sub\u003e receptor (GABA\u003csub\u003eC\u003c/sub\u003e-R). Invertebrate iGABARs do not readily fit the vertebrate GABA\u003csub\u003eA\u003c/sub\u003e/GABA\u003csub\u003eC\u003c/sub\u003e receptor categories (Sieghart 1995). The majority of insect iGABARs are distinguished from vertebrate GABA\u003csub\u003eA\u003c/sub\u003e receptors by their insensitivity to bicuculline and differ from GABA\u003csub\u003eC\u003c/sub\u003e-Rs in that they are subject to allosteric modulation, albeit weakly, by benzodiazepines and barbiturates (Hosie \u003cem\u003eet al\u003c/em\u003e. 1997).\u003c/p\u003e\r\n\r\n\u003cp\u003eChemical interactions with iGABARs can cause a variety of pharmacological and neurotoxicological effects depending on the location of the active or allosteric site affected. Three distinct types of interactions at binding sites on iGABARs can antagonize the postsynaptic inhibitory functions of GABA and lead to epileptic seizures and death. These three types of interactions correspond to three AOPs (Gong \u003cem\u003eet al\u003c/em\u003e. 2015). One of the three types of\u0026nbsp;interaction is non-competitive channel blocking at the picrotoxin convulsant site located inside of the iGABAR pore that spans neuronal cell membranes, which is the MIE described in the current AOP. The other two types of interactions are negative modulation at allosteric sites and competitive binding at the active orthosteric sites (MIEs to be developed in the future).\u003c/p\u003e\r\n\r\n\u003cp\u003eIt is worth noting that there exist another class of GABA receptors called metabotropic G-protein-coupled receptors (mGABAR) or GABA\u003csub\u003eB\u003c/sub\u003e receptors. This AOP is not applicable to GABA\u003csub\u003eB\u003c/sub\u003e receptors because they mediate slow and sustained inhibitory responses to GABA and are involved in absence epilepsy (Han et al. 2012).\u003c/p\u003e\r\n","user_defined_mie":"667: Binding at picrotoxin site, iGABAR chloride channel","user_defined_ao":"613: Occurrence, Epileptic seizure","oecd_project":"1.15","oecd_status_id":1,"graphical_representation_image_uid":"2016/12/02/7xsl9201gi_IGABAR_AOP1.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2019-04-29T12:16:50.000-04:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":8,"handbook_id":1,"project_129":false},{"id":11,"title":"Percellome Toxicogenomics Approach for AOP Building: Case study on Pentachlorophenol","short_name":"Pentachlorophenol Acute Response by Percellome","corresponding_author_id":176,"abstract":"\u003cp\u003eThis case study aims at making AOPs triggered by oral exposure to Pentachlorophenol (PCP) of liver and possible new aspect of systemic toxicity. In this case study, Pentachlorophenol (PCP) was monitored for adult mouse liver transcriptome responses 2, 4, 8 and 24 hours after single oral administration with four dose levels, 0, 10, 30 and 100mg/kg, using Affymetrix GeneChip MOE430 2.0. The expression data were absolutized by the Percellome method and expressed as three dimensional (3D) surface graphs with axises of time, dose and copy numbers of mRNA per cell. Homemade software RSort was used for comprehensive screening of the 3D surface data followed by visual inspection to confirm the significant responses, and PercellomeExploror for cross-referencing. In the first 8 hours, approximately 100 probe sets (PSs) related to PXR/SXR and Cyp2a4 and other metabolic enzymes were induced, and Fos and Junb were suppressed. At 24 hours, about 1,200 PSs were strongly induced. Cross-referencing the Percellome database consisting of 111 chemicals on liver transcriptome revealed that about half of the PSs belonged to the metabolic pathways including Nrf2-mediated oxidative stress response networks, sharing with some of the 111 chemicals. The other half was interferon signalling network genes (ISG), and was unique to PCP. Toll like receptors and other pattern recognition receptors, interferon regulatory factors and interferon alpha itself were included. On the other hand, inflammatory cytokines were not induced. In summary, functional symptoms of PCP, such as hyperthermia and profuse sweating might be mediated by the ISG rather than the hitherto documented mitochondrial uncoupling mechanism. Reports of imiquimod and RO8191 as agonists of toll-like receptor and interferon receptor might associate PCP with a seed for interferon mimetic drugs (cf. Figure below).\u003c/p\u003e\r\n\r\n\u003cp\u003eThis case study will also serve as an example of how the Percellome Project Database and Percellome Analytical Tools can be effectively applied to the AOP development. Further studies including in silico data mining will expand the AOP to cover the chronic liver toxicity induced by PCP.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":4,"authors":"\u003cp\u003eJun Kanno Division of Cellular \u0026amp; Molecular Toxicology, Biological Safety Research Center, National Institute of Health Sciences\u003c/p\u003e\r\n\r\n\u003cp\u003e1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan +81-3-3700-9619 kanno@nihs.go.jp\u003c/p\u003e\r\n","applicability_of_the_aop":"","key_event_essentiality":"","weight_of_evidence_summary":"","quantitative_considerations":"","optional_considerations":"","references":"","overall_assessment":"","background":"","user_defined_mie":"245: Activation, PXR/SXR","user_defined_ao":"","oecd_project":"2.5","oecd_status_id":4,"graphical_representation_image_uid":null,"saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":null,"development_strategy":null,"known_modulating_factors":null,"assigned_license_id":9,"handbook_id":1,"project_129":false},{"id":12,"title":"Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development leads to neurodegeneration with impairment in learning and memory in aging","short_name":"Binding of antagonist to NMDARs can lead to neuroinflammation and neurodegeneration","corresponding_author_id":125,"abstract":"\u003cp\u003eThis AOP is an extension of AOP 13 linking NMDAR chronic inhibition during brain development to impairment of learning and memory. It links chronic NMDA receptors inhibition during brain development to Adverse Outcomes, i.e. neurodegeneration in hippocampus and cortex with amyloid plaque deposition and tau hyperphosphorylation and impairment of learning and memory, which are considered as hallmark of Alzheimer\u0026#39;s disease. It introduces another KE, Neuroinflammation, which is involved in several neurodegenerative diseases. With Neuroinflammation and Neurodegeneration, this AOP connects to AOP 48, where in adult brain, \u0026laquo;\u0026nbsp;neuroinflammation\u0026nbsp;\u0026raquo; leads to \u0026laquo;\u0026nbsp;Neurodegeneration\u0026nbsp;\u0026raquo;\u0026nbsp;; \u0026laquo;\u0026nbsp;Neurodegeneration\u0026nbsp;\u0026raquo; leads to \u0026laquo;\u0026nbsp;Decreased neuronal network function\u0026nbsp;\u0026raquo;, which finally leads to \u0026laquo;\u0026nbsp;Impairement of learning and memory\u0026nbsp;\u0026raquo;. Both neurodegeneration and cognitive deficits are observed in Alzheimer\u0026rsquo;s pathology. But as neurodegenerative diseases are complex and multifactorial, the authors proposed two Adverse outcomes: one at the organism level \u0026laquo;\u0026nbsp;Impairment of learning and memory\u0026raquo;, and one at the organ level, \u0026laquo;\u0026nbsp;neurodegeneration\u0026nbsp;\u0026raquo;. Both are regulatory endpoints. This AOP integrates in the network of AOPs relative to neurotoxicity testing.\u003c/p\u003e\r\n\r\n\u003cp\u003eThis AOP is based on the hypothesis of Landrigan and coworkers (2005) proposing an early origin of neurodegenerative diseases in later life. The chemical initiator known to block NMDARs and used in this AOP for the empirical support is lead (Pb), which is a well-known developmental neurotoxicant. In epidemiological studies of adults, cumulative lifetime lead exposure has been associated with accelerated decline in cognition (Bakulski et al., 2012), suggesting that long term exposure to lead during brain development or occupational exposure in adulthood increases the risk to develop a neurodegenerative disease of Alzheimer\u0026#39;s type. The long latency period between exposure and late-onset of neurodegeneration and cognitive deficits gives a very broad life-stage applicability, where developmental exposure has consequences in the aging brain. Such a long temporal delay between exposure and adverse outcome is a real difficulty and challenge for neurotoxicity testing. As the Key Event \u0026laquo;\u0026nbsp;Neuroinflammation\u0026nbsp;\u0026raquo; appears to play a crucial role in the neurodegenerative process, the authors propose to include the measurement of this apical KE in the battery of regulation-required neurotoxicity testing.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":1,"authors":"\u003cp\u003eFlorianne Tschudi-Monnet, Department of Physiology, University of Lausanne, Switzerland, and Swiss Center for Applied Human Toxicology (SCAHT), Florianne.Tschudi-Monnet@unil.ch, corresponding author\u003c/p\u003e\r\n\r\n\u003cp\u003eRex FitzGerald, SCAHT, Universit\u0026auml;t Basel, Missionsstrasse 64, CH-4055 Basel, Rex.FitzGerald@unibas.ch\u003c/p\u003e\r\n\r\n\u003cp\u003eAcknowledgments: The authors greatly acknowledged the contribution of Drs Anna Price and Magda Sachana who prepared the MIE and KE1-KE4 as well as the related KERs of this AOP.\u003c/p\u003e\r\n\r\n\u003cp\u003eAnna Price, Joint Research Centre Institute for Health and Consumer Protection Systems Toxicology Unit Via E. Fermi 2749 - 21020 - Ispra (VA) -Italy, e-mail address: PRICE Anna \u0026lt;Anna.PRICE@ec.europa.eu\u0026gt;\u003c/p\u003e\r\n\r\n\u003cp\u003eMagdalini Sachana, Joint Research Centre Institute for Health and Consumer Protection Systems Toxicology Unit Via E. Fermi 2749 - 21020 - Ispra (VA) -Italy, present e-mail address: \u0026quot;Magdalini.SACHANA@oecd.org\u0026quot; \u0026lt;Magdalini.SACHANA@oecd.org\u0026gt;\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003eThis AOP is not sex dependent. Regarding the life stage applicability, MIE induced during brain development can have consequences when brain is aging, according to the hypothesis proposed by Landrigan and coworkers (2005). However, it is also possible that the AO does not depend exclusively on developmental exposure, since cumulative occupational exposure also decreased cognitive functions in aging (Stewart et al., 2006).\u003c/p\u003e\r\n","key_event_essentiality":"\u003cp\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eTable: Essentiality of KEs\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cdiv\u003e\r\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" class=\"MsoTableGrid\" style=\"border-collapse:collapse; border:none; margin-left:-30.05pt; width:758px\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd rowspan=\"2\" style=\"width:158.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003e2 Support for Essentiality of KEs\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd rowspan=\"2\" style=\"width:124.95pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eDefining Question\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eAre downstream KEs and/or the AO prevented if an upstream KE is blocked\u0026nbsp;?\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:149.4pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eHigh (Strong)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:163.05pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eModerate\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:163.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eLow (Weak)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:149.4pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eDirect evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g. stop/reversibility studies, antagonism, knock out models, etc.)\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:163.05pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eIndirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:163.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eNo or contradictory experimental evidence of the essentiality of any of the KEs\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:158.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eKE1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman\"\u003eNMDARs inhibition \u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:124.95pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eSTRONG\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"width:475.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eActivation of NMDAR results in LTP, which is related to increase synaptic strength and memory formation in hippocampus (Johnston et al., 2009).\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:158.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eKE2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman\"\u003eCalcium influx decreased \u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:124.95pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eSTRONG\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"width:475.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eIn CNS, many intracellular responses to modified calcium level are mediated by calcium/calmoduline-regulated protein kinases (Wayman et al., 2008). Mice with a mutation of calmoduline kinase II, which is abundantly found in hippocampus, have shown spatial learning impairment (Silva et al., 1992)\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:158.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eKE3 \u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman\"\u003eRelease of BDNF, reduced \u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:124.95pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eSTRONG\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"width:475.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eBDNF serves essential function in synaptic plasticity (Poo, 2001) and is crucial for learning and memory processes (Lu et al., 2008). Precursor form of BDNF and mature BDNF are decreased in the preclinical stages of Alzheimer\u0026#39;s disease (Peng et al., 2005)\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:158.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eKE4\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman\"\u003eCell Injury/death, increased\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:124.95pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eSTRONG\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"width:475.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eSeveral studies dealing with postnatal administration of NMDAR antagonists such as MK 801, ketamine or ethanol have shown a devastating cell apoptotic degeneration in several brain areas of animal models resulting in learning deficits (Creeley and Olney, 2013)\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:158.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eKE5 \u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman\"\u003eNeuroinflammation\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:124.95pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eMODERATE\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"width:475.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eRationale: Rats treated with Pb from PND 24 to 80 showed a neuroinflammatory response associated with neuronal death in hippocampus and LTP impairment. These effects were significantly reversed by administration of minocycline, an antibiotic known to block microglial reactivity (Liu et al., 2012), demonstrating the essentiality of neuroinflammation for neurodegeneration in hippocampus and impairment of memory processes. In addition, the fact that neuroinflammation triggered during brain development by a systemic immune challenge caused Alzheimer\u0026#39;s like pathology (Krstic et al., 2012), showed the central role of neuroinflammation in this pathology. In addition, in a mouse model of Alzheimer\u0026#39;s disease, the blockade of microglial cell proliferation and the shifting of the microglial inflammatory profile to an anti-inflammatory phenotype by inhibiting the colony-stimulating factor 1 receptor on microglial cells, prevented synaptic degeneration and improved cognitive functions (Olmos-Alonso et al., 2016). This latter experiment has not been done during brain development. But the hypothesis is that a chronic neuroinflammation during a prolonged period increased the risk to develop an Alzheimer\u0026#39;s neurodegenerative disease in aging (Krstic and Knuesel, 2013). \u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eHowever, as other mechanisms such epigenetic modifications can lead to accumulation of amyloid plaques- and tau hyperphosphorylation-related neurodegeneration, and due to some inconsistencies of anti-inflammatory treatments as protection against the neurodegenerative process, the essentiality of Neuroinflammation was considered as moderate.\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"height:90.55pt; width:158.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eAO (at organ level)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman\"\u003eNeurodegeneration in\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman\"\u003e\u0026nbsp;hippocampus and cortex\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"height:90.55pt; width:124.95pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eSTRONG\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"height:90.55pt; width:475.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eSeveral studies descibed Pb-induced accumulation of amyloid peptides and hyperphosphorylated and Pb-induced cell injury/deathin hippocampus or decrease in hippocampal volume, what are all well accepted landmarks of Alzheimer\u0026#39;s pathology (Lloret et al., 2015). As described in AOP 48, neurodegeneration can lead to \u0026quot;Decreased neuronal network function\u0026quot; which in turn leads to \u0026quot;impairment of learning and memory\u0026quot;, which is also considered as a hallmark of Alzheimer\u0026#39;s pathology (Schoemaker et al., 2014). \u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:10.0pt\"\u003eHowever, there is some controversy about the relationship between increased accumulation of amyloid plaques and increased cognitive deficits: \u0026nbsp;Lichtenstein and coworkers (2010) described that accumulation of amyloid plaques reaches a plateau, whereas a temporal relationship is observed between increased microglial activation, widespread degeneration (decreased hippocampal volume) and increased cognitive deficits. Therefore the essentiality for accumulation of amyloid and tau to cognitive deficits should be considered as moderate. But, as cell injury/death in hippocampus and cortex or decrease in hippocampal volume due to widespread neurodegeneration is strongly associated to impairment in learning and memory, the essentiality of this KE has been rated as strong.\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:158.0pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003e\u0026nbsp;\u003cstrong\u003eAO (at organism level)\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-family:times new roman\"\u003eImpairment of learning and memory \u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:124.95pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003eSTRONG\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"width:475.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:justify\"\u003eNeurodegenerative diseases are complex and multifactorial, depend on gene-environment interactions, and have a slow temporal evolution (Sherer et al., 2002; Steece-Collier et al., 2002; Tsang and Soong, 2003); Mutter et al., 2004). A direct association between Pb exposure during brain development and Alzheimer\u0026#39;s pathology is not supported by epidemiological studies. However, two studies reported that past adult exposure is linked with neurodegeneration (Stewart et al., 2006) and decline in cognitive function (Schwartz et al., 2000), effects which were observed long after exposure ceases. Tibia lead levels were good predictors of these delayed effects. Another study showed an association between lead exposure early in life with cognitive and behavioral consequences in early adulthood (Agency for toxic substances, 1997). Despite the lack of specific epidemiological evidence, the principle of delayed effects occuring long after exposure, as well as strong evidence from experimental studies (for review, see Chin-Chan et al., 2015) suggest that long-term exposure to environmental toxicants such as Pb during brain development or exposure later in life can be considered as a risk factor for the development of neurodegenerative diseases in aging.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:times new roman; font-size:12.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\u003c/div\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003e\u003cstrong\u003e1. Concordance of dose-response and temporal concordance between KEs and the AO\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eIt is difficult to analyze the dose-response relationships between the different KEs, (i) because of the long temporal delay between MIE and AOs\u0026nbsp;; (ii) because no study has analyzed them simultaneously, and (iii) \u0026nbsp;because of the difficulties in extrapolating \u003cem\u003ein vitro\u003c/em\u003e to \u003cem\u003ein vivo\u003c/em\u003e data. As the apical KEs and AO occur and can be measured years after exposure, even when Pb blood level has returned to normal, measurement of bone Pb content has been proposed as a measurement of historical Pb exposure in adults (Bakulski et al., 2012, 2014).\u0026nbsp; The following table gives an overview of the doses/concentrations and exposure duration at which the different KEs were measured.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" class=\"MsoTableGrid\" style=\"border-collapse:collapse; border:none; margin-left:4.05pt; width:535.8pt\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:66.35pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eKE1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:72.1pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eKE2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:53.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eKE3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:68.1pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eKE4\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:92.65pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eK5\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:104.35pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eAO at organ level\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:79.05pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eAO at organism level\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:66.35pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eNMDAR inhibition\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:72.1pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eCalcium influx, decreased\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:53.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eBDNF release, decreased\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:68.1pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eCell injury/death \u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:92.65pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eNeuroinflammation\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:104.35pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eNeurodegeneration with amyloid plaques and tau hyperphosphorylation\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:79.05pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003eImpairment of learning and memory\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:66.35pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb 2.5-5 \u003c/span\u003e\u003cspan style=\"font-family:symbol; font-size:9.0pt\"\u003em\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003eM acute\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003einhibits\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eNMDAR whole cell and channel current in hippocampal neurons \u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Alkondon et al., 1990)\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:72.1pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb 100 nM 1h-24h\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003edecrease Ca2+ in embryonic rat hippocampal neurons \u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Ferguson et al., 2000)\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:53.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eNo direct evidence\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:68.1pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb 2mM in drinking water 3 weeks before mating till weaning (PND 21) resulting in\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eat PND 21 \u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb blood 108.8 \u003c/span\u003e\u003cspan style=\"font-family:symbol; font-size:9.0pt\"\u003em\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003eg/L\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb hippoc. 0.253 \u003c/span\u003e\u003cspan style=\"font-family:symbol; font-size:9.0pt\"\u003em\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003eg/g\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eat PND 91\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb blood 39.27 \u003c/span\u003e\u003cspan style=\"font-family:symbol; font-size:9.0pt\"\u003em\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003eg/L\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb hippoc. 0.196 \u003c/span\u003e\u003cspan style=\"font-family:symbol; font-size:9.0pt\"\u003em\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003eg/g\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eabout 35% decrease in synapses in hippocampus\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eabout 30% decrease of hippocampal neurons\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Xiao et al., 2014)\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:92.65pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eIn vivo\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e0.22 ppm (together with As and Cd) from gestational day 5 till day 180\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ein adulthood: IL-1\u003c/span\u003e\u003cspan style=\"font-family:symbol; font-size:9.0pt\"\u003eb\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e, TNF-\u003c/span\u003e\u003cspan style=\"font-family:symbol; font-size:9.0pt\"\u003ea\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e, IL-6 increased 2x\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eAshok et al., 2015\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eRats exposed to Pb 100 ppm for 8 weeks (from PND 24 to 80) caused at the end of treatment microglial activation in hippocampus. \u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Liu et al., 2012\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eIn vitro\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e10\u003csup\u003e-6\u003c/sup\u003e-10\u003csup\u003e-4\u003c/sup\u003e M for 10 days\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ein 3D cultures of fetal rat brain cells\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003emicroglial and astrocyte reactivities\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Zurich et al., 2002)\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eco-cultures of hippocampal neurons with microglial cells treated with Pb (50 micomol/L for 48h) caused microglial activation and upregulation of IL-1beta, TNF-alpha and i_NOS\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Liu et al., 2012)\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:104.35pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eMonkeys exposed to\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb 1.5 mg/kg/day\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003efrom birth to 400 days\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eat 23 years of age\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eTau accumulation\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eOverexpression of amyloid-beta protein precursor and of amyloid-beta\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eenhanced pathologic neurodegeneration\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Bihaqi et al., 2011; Bihaqi and Zawia, 2013)\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eMice exposed to\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb 0.2% in drinking water from PND 1-20 or from PND 1-20 + From 3-7 months\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eat 700 days of age\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eelevated protein and mRNA for tau\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eand\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eaberrant site-specific tau hyperphosphorylation\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Bihaqi et al., 2014)\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eHuman Tg-SWDI APP transgenic mice , PB 50 mg/kg by gavage for 6 weeks exhibit increase AB in CSF, cortex and hippocampus and increased amyloid plaque load (Gu et al., 2012)\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eRats exposed to Pb 100 ppm for 8 weeks (from PND 24 to 80) caused at the end of treatment neuronal death in hippocampus. \u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Liu et al., 2012)\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:79.05pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eMice exposed to\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003ePb 0.2% in drinking water from PND 1-20 or from PND 1-20 and from 3-7 months\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eTested at 700 days of age\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eDecrease in cognitive functions (Morris water maze, Y maze testing for spatial memory and memory, a hippocampal formation-dependent task)\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Bihaqi et al., 2014b)\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eRats exposed to Pb 100 ppm for 8 weeks (from PND 24 to 80) reduced hippocampal LTP level at the end of the treatment \u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e(Liu et al., 2012)\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eHuman Tg-SWDI APP transgenic mice , PB 50 mg/kg by gavage for 6 weeks showed an impaired spatial learning (Gu et al., 2012)\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n\u003cstrong\u003e2. Strength, consistency and association of AO and MIE\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThe accepted molecular mechanism of action of the chemical initiator Pb is inhibition of NMDARs (Alkondon et al., 1990; Gavazzo et al., 2001, 2008; Guilarte et al., 1992; Omelchenko et al., 1997) and several experimental studies in rat, monkey and zebrafish linked chronic exposure to Pb during brain development to Alzheimer\u0026#39;s-like neurodegeneration with cognitive deficits (Zawia and Basha, 2005; Basha and Reddy, 2010; Bihaqi et al., 2014a; Bihaqi et al., 2014 b; Lee and Freeman, 2014). This AOP is defined by a single environmental chemical, Pb. However, other NMDAR antagonists used as general anesthetics (MK 801, phenylcyclidine, ketamine) applied during brain development may also lead to functional impairments in cognitive domains relevant to memory. The effects of these anesthetics on brain function appear to have a delayed onset, and can be very long-lasting if not permanent. In general, longer durations, higher concentrations and longer or repeated exposures tend to exacerbate impairments (for review, see Walters and Paule, 2017). The mechanisms underlying anesthetic-induced neurotoxicity are unclear, but several hypotheses have been proposed: impairment of mitochondrial integrity and function, dysregulation of intracellular calcium and neuroinflammation have all been implicated (Lei et al., 2012). Some of these mechanisms are common to the KEs described in this AOP, suggesting that such delayed effects on memory processes can be a general consequence of developmental brain exposure to NMDAR inhibitors. However, no studies have yet reported that these other NMDAR inhibitors cause amyloid plaque deposition or tau hyperphosphorylation associated with Alzheimer-like neurodegeneration when aging.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eInterestingly, memantine, a NMDAR antagonist used in the treatment of Alzheimer\u0026#39;s disease, was shown to improve cognitive functions (for review, see Dekundy, 2006). This might be considered as a discrepancy with the described AOP considering Pb as an antagonist of NMDAR and its potential risk to cause cognitive deficits and amyloid plaque accumulation, which are hallmarks of Alzheimer\u0026#39;s disease. However, memantine antagonism of NMDAR is quite different (low affinity and voltage-dependent) and the window of exposure differs completely, since memantine is applied in aged patients when the disease has broken out; whereas the risk of delayed neurodegeneration described in this AOP is due to NMDAR inhibition during brain development.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n\u003cstrong\u003e3. Biological Plausibility, and empirical support\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cdiv\u003e\r\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" class=\"MsoTableGrid\" style=\"border-collapse:collapse; border:none; margin-left:4.05pt; width:535.8pt\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman\"\u003eDefining Question\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eHigh /Strong\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eModerate\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003eLow/weak\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cstrong\u003e\u003cspan style=\"font-family:times new roman\"\u003eSupport for Biological Plausibility of KERs\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-family:times new roman; font-size:9.0pt\"\u003eIs there a mechanistic (i.e. structural or functional) relationship between KEup and KEdown consistent with established biological knowledge?\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-family:times new roman; font-size:9.0pt\"\u003eExtensive understanding of the KER based on extensive previous documentation and broad acceptance\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-family:times new roman; font-size:9.0pt\"\u003eThe KER is plausible based on analogy to accept biological relationship but scientific understanding is not completely established\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-family:times new roman; font-size:9.0pt\"\u003eThere is empirical support for a statistical association between KEs but the structural or functional relationship between them is not understood\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eMIE to KE inhibition of NMDARs\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eExtensive understanding\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eLimited conflicting data\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eKE NMDAR inhibition to KE calcium influx, decreased\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eExtensive understanding\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eLimitied conflicting data\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eKE calcium influx, decreased to KE release of BDNF, decreased\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eExtensive understanding\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eLimited conflicting data\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eKE release of BDNF, decreased to KE Cell \u0026nbsp;Injury/death\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eExtensive understanding\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eLimited conflicting data\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eKE Cell injury/death to KE Neuroinflammation \u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eThe general mechanisms linking cell injury/death to neuroinflammation is well accepted. However, it is mainly descibed in adult brain. However, a neuroinflammatory response was found following Pb exposure of 3D cultures during synaptogenesis and myelination (Zurich et al., 2002). A controversy exists about apoptosis and neuroinflammation, but some empirical evidences has been provided.\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eThe fact that cell injury/deat leads to neuroinflammation and that neuroinflammation leads to neurodegeneration is known as avicious circle and is involved in neurodegenerative diseases, suggesting that neuroinflammation exacerbates the neurodegenerative process (Griffin et al., 1998; 2006)\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eKE Neuroinflammation to AO Neurodegeneration in Hippocampus and cortex\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eIn adult, the early involvement of neuroinflammation in the neurodegenerative process is widely accepted.\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eIn immature brain, one study in mice link gestational induction of neuroinflammation to late neurodegeneration with accumulation of aberrant amyloid and tau (Kristic et al., 2012).\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eThere is in vitro experimental data following Pb exposure linking neuroinflammation to extensive neuronal death in immature cells.\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eIn vivo, There are several studies linking early Pb exposure to late neurodegeneration in several species. However, the mechanisms involved is epignenetic modifications of genes involved in the amyloid cascade. Such epigenetic modifications may be due to ROS released by the neuroinflammatory process (Bolin et al., 2006).\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003eTherefore the link may be indirect and needs further analyses.\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003eAO Neurodegeneration in hippocampus and cortex to KE Neuroinflammation\u0026nbsp;\u003cspan style=\"font-size:9.0pt\"\u003e \u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003eConcept of vicious circle where neuroinflammation lead to neurodegeneration and vice versa (Griffin et al., 1998, 2006)\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003eThere are no specific empirical data for the chemical initiator Pb.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"width:97.55pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;AO Neurodegeneration in hippocampus and cortex\u0026nbsp; to AO Impairment of learning and memory\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:120.5pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:118.45pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;The role of hippocampus in memory processes is well accepted. Alterations of LTP in hippocampus of rats exposed to Pb has been described (Liu et al., 2012), as well as preferential accumulation of hyperphosphorylated tau in frontal cortex of mice exposed during development to Pb. These mice exhibited cognitive deficit when aging (Bihaqi et al., 2014b).\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"width:112.2pt\"\u003e\r\n\t\t\t\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\u003c/div\u003e\r\n","quantitative_considerations":"\u003cp\u003eWith an Adverse Outcome occurring after such a long delay after the MIE, it is extremely difficult to make a quantitative link, since the AO can occur when serum Pb levels have returned to normal. Bakulski and coworkers (2012) therefore proposed measuring Pb bone content as an index of historical Pb exposure. Similarly, Schwartz and coworkers (2000) showed that tibia Pb levels were good predictors of delayed cognitive decline of former organolead workers. Thus, Pb blood level is rather representative of acute exposure, whereas Pb bone level represents long-term accumulation (Dorsey et al., 2006).\u003c/p\u003e\r\n\r\n\u003cp\u003eRegarding the KER \u0026quot;cell injury/death to neuroinflammation\u0026quot;, it is accepted that neuronal injury may be sufficient to trigger a neuroinflammatory response. But, because of the neuroprotective or neuroreparative potential of neuroinflammation, it is possible that the consequences of neuroinflammation will be in a first step positive, with microglia expressing the M2 phenotype. After an exposure arrest and a temporal delay (Sandstr\u0026ouml;m et al., 2014), or in the presence of cell death (Nakajima and Kohsaka, 2004; Hanish and Kettenmann, 2007), microglia can acquire the M1 neurodegenerative phenotype. Therefore, it is rather the qualitative phenotype of neuroinflammation that will induce neurodegeneration. However, a possible correlation of increased microglial reactivity, measured by PET, and a decrease in hippocampal volume, measured by MRI, suggests, in advanced Alzheimer\u0026#39;s disease, a possible link between the intensity of neuroinflammation and the neurodegenerative consequences (Lichtenstein et al., 2010).\u003c/p\u003e\r\n","optional_considerations":"\u003cp\u003eThis AOP aims at giving a conceptual framework to mechanistically understand an apical hazard, which can occur long after initial exposure; this hazard is not captured in standard regulatory neurotoxicity testing.\u003c/p\u003e\r\n\r\n\u003cp\u003eThe KE \u0026quot;neuroinflammation\u0026quot;, which is shared with other AOPs, appears to play an early and central role in the neurodegenerative process (Eikelenboom et al., 2000; Whitton, 2007; Krstic et al., 2012). Neuroinflammation is observed in most neurodegenerative diseases including Alzheimer\u0026#39;s disease \u0026nbsp;(Whitton, 2007\u0026nbsp;; Tansey and Goldberg, 2009\u0026nbsp;; Niranjan, 2014\u0026nbsp;; \u003cspan style=\"font-family:times new roman; font-size:11.0pt\"\u003eVerkhratsky\u003c/span\u003e et al., 2014). Neuroinflammation can also be triggered by several classes of toxicants (Monnet-Tschudi et al., 2007). Any toxicant able to trigger a neuroinflammatory response expressing the neurodegenerative phenotype should be considered as a risk factor for neurodegenerative diseases. Therefore, testing for toxicant-induced neuroinflammation should be used as an endpoint in regulatory toxicology. The standard neurotoxicity testing does not require measurement of any marker of neuroinflammation, except for fuel additives, where testing for a potential increase in glial fibrillary acidic protein (GFAP), as marker of astrocyte reactivity, is mandatory according to US EPA (40 CFR 79 67).\u003c/p\u003e\r\n\r\n\u003cp\u003eThe evolution of regulation towards mechanistically-driven approaches for supporting hazard identification implies also the development of \u003cem\u003ein vitro \u003c/em\u003etesting. Three-dimensional cultures, prepared from fetal rat brain cells, exhibiting an histotypic organisation comprising all types of brain cells (specifically microglial cells and astrocytes, as effector cells of neuroinflammation) and allowing long-term maintenance for repeated exposure and for studying the evolution of neuroinflammatory phenotypes, are already available (Al\u0026eacute;p\u0026eacute;e et al., 2014; Monnet-Tschudi et al., 2007\u0026nbsp;; Sandstr\u0026ouml;m et al., 2014). Similar 3D cultures prepared from human pluripotent stem cells are in development (Schwartz et al., 2015; Sandstr\u0026ouml;m et al., 2017).\u003c/p\u003e\r\n","references":"\u003cdiv\u003e\r\n\u003cp\u003eATSDR 2007. Toxicological Profile for Lead. U.S. Agency for Toxic Substances and Disease Registry, August 2007.\u003c/p\u003e\r\n\r\n\u003cp\u003eAl\u0026eacute;p\u0026eacute;e N, Bahinski A, Daneshian M, De Wever B, Fritsche E, Goldberg A, et al. 2014. State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology. Altex 31(4): 441-477.\u003c/p\u003e\r\n\r\n\u003cp\u003eAlkondon M, Costa AC, Radhakrishnan V, Aronstam RS, Albuquerque EX. 1990. Selective blockade of NMDA-activated channel currents may be implicated in learning deficits caused by lead. FEBS Lett 261(1): 124-130.\u003c/p\u003e\r\n\r\n\u003cp\u003eAschner M, Ceccatelli S, Daneshian M, Fritsche E, Hasiwa N, Hartung T, et al. 2017. Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: example lists and criteria for their selection and use. Altex 34(1): 49-74.\u003c/p\u003e\r\n\r\n\u003cp\u003eAshok A, Rai NK, Tripathi S, Bandyopadhyay S. 2015. Exposure to As-, Cd-, and Pb-mixture induces Abeta, amyloidogenic APP processing and cognitive impairments via oxidative stress-dependent neuroinflammation in young rats. Toxicol Sci 143(1): 64-80.\u003c/p\u003e\r\n\r\n\u003cp\u003eBakulski KM, Rozek LS, Dolinoy DC, Paulson HL, Hu H. 2012. Alzheimer\u0026#39;s disease and environmental exposure to lead: the epidemiologic evidence and potential role of epigenetics. Curr Alzheimer Res 9(5): 563-573.\u003c/p\u003e\r\n\r\n\u003cp\u003eBakulski, K.M., Park, S.K., Weisskopf, M.G., Tucker, K.L., Sparrow, D., Spiro, A., 3rd, Vokonas, P.S., Nie, L.H., Hu, H., Weuve, J., 2014. Lead exposure, B vitamins, and plasma homocysteine in men 55 years of age and older: the VA normative aging study. Environ Health Perspect 122(10), 1066-1074.\u003c/p\u003e\r\n\r\n\u003cp\u003eBasha R, Reddy GR. 2010. Developmental exposure to lead and late life abnormalities of nervous system. Indian journal of experimental biology 48(7): 636-641.\u003c/p\u003e\r\n\r\n\u003cp\u003eBihaqi SW, Huang H, Wu J, Zawia NH. 2011. Infant exposure to lead (Pb) and epigenetic modifications in the aging primate brain: implications for Alzheimer\u0026#39;s disease. J Alzheimers Dis 27(4): 819-833.\u003c/p\u003e\r\n\r\n\u003cp\u003eBihaqi SW, Zawia NH. 2013. Enhanced taupathy and AD-like pathology in aged primate brains decades after infantile exposure to lead (Pb). Neurotoxicology 39: 95-101.\u003c/p\u003e\r\n\r\n\u003cp\u003eBihaqi SW, Bahmani A, Adem A, Zawia NH. 2014a. Infantile postnatal exposure to lead (Pb) enhances tau expression in the cerebral cortex of aged mice: relevance to AD. Neurotoxicology 44: 114-120.\u003c/p\u003e\r\n\r\n\u003cp\u003eBihaqi SW, Bahmani A, Subaiea GM, Zawia NH. 2014b. Infantile exposure to lead and late-age cognitive decline: relevance to AD. Alzheimer\u0026#39;s \u0026amp; dementia: the journal of the Alzheimer\u0026#39;s Association 10(2): 187-195.\u003c/p\u003e\r\n\r\n\u003cp\u003eBolin CM, Basha R, Cox D, Zawia NH, Maloney B, Lahiri DK, et al. 2006. Exposure to lead and the developmental origin of oxidative DNA damage in the aging brain. Faseb J 20(6): 788-790.\u003c/p\u003e\r\n\r\n\u003cp\u003eChin-Chan, M., Navarro-Yepes, J., Quintanilla-Vega, B., 2015. Environmental pollutants as risk factors for neurodegenerative disorders: Alzheimer and Parkinson diseases. Front Cell Neurosci 9, 124.\u003c/p\u003e\r\n\r\n\u003cp\u003eCho SH, Chen JA, Sayed F, Ward ME, Gao F, Nguyen TA, et al. 2015. SIRT1 deficiency in microglia contributes to cognitive decline in aging and neurodegeneration via epigenetic regulation of IL-1beta. J Neurosci 35(2): 807-818.\u003c/p\u003e\r\n\r\n\u003cp\u003eCreeley CE, Olney JW. (2013) Drug-Induced Apoptosis: Mechanism by which Alcohol and Many Other Drugs can Disrupt Brain Development. Brain Sci. 3: 1153\u0026ndash;1181.\u003c/p\u003e\r\n\r\n\u003cp\u003eDekundy A.2006. Coadministration of memantine with Acetylcholinesterase Inhibitors: Preclinical and Clinical Evidence. In: Toxicology of Organophosphate and Carbamate Compounds. Gupta RC, editor. Elsevier. Amsterdam. Chap. 4. pp 35-46.\u003c/p\u003e\r\n\r\n\u003cp\u003eDorsey CD, Lee BK, Bolla KI, Weaver VM, Lee SS, Lee GS, et al. 2006. Comparison of patella lead with blood lead and tibia lead and their associations with neurobehavioral test scores. Journal of occupational and environmental medicine / American College of Occupational and Environmental Medicine 48(5): 489-496.\u003c/p\u003e\r\n\r\n\u003cp\u003eEikelenboom P, Rozemuller AJ, Hoozemans JJ, Veerhuis R, van Gool WA. 2000. Neuroinflammation and Alzheimer disease: clinical and therapeutic implications. Alzheimer Dis Assoc Disord 14 Suppl 1: S54-61.\u003c/p\u003e\r\n\r\n\u003cp\u003eFerguson C, Kern M, Audesirk G. 2000. Nanomolar concentrations of inorganic lead increase Ca2+ efflux and decrease intracellular free Ca2+ ion concentrations in cultured rat hippocampal neurons by a calmodulin-dependent mechanism. Neurotoxicology 21(3): 365-378.Gavazzo P, Gazzoli A, Mazzolini M, Marchetti C. 2001. Lead inhibition of NMDA channels in native and recombinant receptors. Neuroreport 12(14): 3121-3125.\u003c/p\u003e\r\n\r\n\u003cp\u003eGavazzo P, Zanardi I, Baranowska-Bosiacka I, Marchetti C. 2008. Molecular determinants of Pb2+ interaction with NMDA receptor channels. Neurochem Int 52(1-2): 329-337.\u003c/p\u003e\r\n\r\n\u003cp\u003eGriffin WST, Sheng JG, Royston MC, Gentleman SM, McKenzie JE, Graham DI, et al. 1998. Glial-neuronal interactions in Alzheimer\u0026#39;s disease:\u0026nbsp; The potential role of a \u0026#39;cytokine cycle\u0026#39; in disease progression. Brain Pathol 8: 65-72.\u003c/p\u003e\r\n\r\n\u003cp\u003eGriffin WS. 2006. Inflammation and neurodegenerative diseases. Am J Clin Nutr 83(2): 470S-474S.\u003c/p\u003e\r\n\r\n\u003cp\u003eGu H, Robison G, Hong L, Barrea R, Wei X, Farlow MR, et al. 2012. Increased beta-amyloid deposition in Tg-SWDI transgenic mouse brain following \u003cem\u003ein vivo\u003c/em\u003e lead exposure. Toxicol Lett 213(2): 211-219.\u003c/p\u003e\r\n\r\n\u003cp\u003eGuilarte TR, Miceli RC. 1992. Age-dependent effects of lead on [3H]MK-801 binding to the NMDA receptor-gated ionophore: \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies. Neurosci Lett 148(1-2): 27-30.\u003c/p\u003e\r\n\r\n\u003cp\u003eHanisch UK, Kettenmann H. 2007. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10(11): 1387-1394.\u003c/p\u003e\r\n\r\n\u003cp\u003eJohnstone AFM, Gross GW, Weiss D, Schroeder O, Shafer TJ. 20. Use of microelectrode arrays for neurotoxicity testing in the 21st century Neurotoxicology 31: 331-350.\u003c/p\u003e\r\n\r\n\u003cp\u003eKrstic D, Madhusudan A, Doehner J, Vogel P, Notter T, Imhof C, Manalastas A, Hilfiker M, Pfister S, Schwerdel C, et al. 2012. Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice. J Neuroinflammation 9:151.\u003c/p\u003e\r\n\r\n\u003cp\u003eKrstic D, Knuesel I: Deciphering the mechanism underlying late-onset Alzheimer disease. Nat Rev Neurol 2013, 9:25-34.\u003c/p\u003e\r\n\r\n\u003cp\u003eLandrigan PJ, Sonawane B, Butler RN, Trasande L, Callan R, Droller D. 2005. Early environmental origins of neurodegenerative disease in later life. Environ Health Perspect 113(9): 1230-1233.\u003c/p\u003e\r\n\r\n\u003cp\u003eLee J, Freeman JL. 2014. Zebrafish as a model for investigating developmental lead (Pb) neurotoxicity as a risk factor in adult neurodegenerative disease: a mini-review. Neurotoxicology 43: 57-64.\u003c/p\u003e\r\n\r\n\u003cp\u003eLei X, Guo Q, Zhang J. 2012. Mechanistic insights into neurotoxicity induced by anesthetics in the developing brain. International journal of molecular sciences 13(6): 6772-6799.\u003c/p\u003e\r\n\r\n\u003cp\u003eLichtenstein MP, Carriba P, Masgrau R, Pujol A, Galea E. 2010. Staging anti-inflammatory therapy in Alzheimer\u0026#39;s disease. Frontiers in aging neuroscience 2: 142.\u003c/p\u003e\r\n\r\n\u003cp\u003eLiu, M.C., Liu, X.Q., Wang, W., Shen, X.F., Che, H.L., Guo, Y.Y., Zhao, M.G., Chen, J.Y., Luo, W.J., 2012. Involvement of microglia activation in the lead induced long-term potentiation impairment. PLoS One 7(8), e43924.\u003c/p\u003e\r\n\r\n\u003cp\u003eLloret, A., Fuchsberger, T., Giraldo, E., Vina, J., 2015. Molecular mechanisms linking amyloid beta toxicity and Tau hyperphosphorylation in Alzheimers disease. Free Radic Biol Med 83, 186-191.\u003c/p\u003e\r\n\r\n\u003cp\u003eMonnet-Tschudi F, Zurich MG, Honegger P. 2007. Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 26(4): 339-346.\u003c/p\u003e\r\n\r\n\u003cp\u003eMutter, J., Naumann, J., Sadaghiani, C., Schneider, R., Walach, H., 2004. Alzheimer disease: mercury as pathogenetic factor and apolipoprotein E as a moderator. Neuro Endocrinol Lett 25(5), 331-339.\u003c/p\u003e\r\n\r\n\u003cp\u003eNakajima K, Kohsaka S. 2004. Microglia: Neuroprotective and neurotrophic cells in the central nervous system. Current Drug Targets-Cardiovasc \u0026amp; Haematol Disorders 4: 65-84.\u003c/p\u003e\r\n\r\n\u003cp\u003eNiranjan R. 2014. The role of inflammatory and oxidative stress mechanisms in the pathogenesis of Parkinson\u0026#39;s disease: focus on astrocytes. Mol Neurobiol 49(1): 28-38.\u003c/p\u003e\r\n\r\n\u003cp\u003eOlmos-Alonso, A., Schetters, S.T., Sri, S., Askew, K., Mancuso, R., Vargas-Caballero, M., Holscher, C., Perry, V.H., Gomez-Nicola, D., 2016. Pharmacological targeting of CSF1R inhibits microglial proliferation and prevents the progression of Alzheimer\u0026#39;s-like pathology. Brain 139(Pt 3), 891-907.\u003c/p\u003e\r\n\r\n\u003cp\u003eOmelchenko IA, Nelson CS, Allen CN. 1997. Lead inhibition of N-methyl-D-aspartate receptors containing NR2A, NR2C and NR2D subunits. J Pharmacol Exp Ther 282(3): 1458-1464.\u003c/p\u003e\r\n\r\n\u003cp\u003ePeng, S., Wuu, J., Mufson, E.J., Fahnestock, M. 2005. Precursor form of brain-derived neurotrophic factor and mature brain-derived neurotrophic factor are decreased in the pre-clinical stages of Alzheimer\u0026#39;s disease. J Neurochem 93(6), 1412-1421.\u003c/p\u003e\r\n\r\n\u003cp\u003ePoo MM. (2001) Neurotrophins as synaptic modulators. Nat Rev Neurosci. 2: 24\u0026ndash;32.\u003c/p\u003e\r\n\r\n\u003cp\u003eSandstr\u0026ouml;m von Tobel, J., D. Zoia, et al. (2014). \u0026quot;Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures.\u0026quot; Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001\u003c/p\u003e\r\n\r\n\u003cp\u003eSandstrom J, Eggermann E, Charvet I, Roux A, Toni N, Greggio C, et al. 2017. Development and characterization of a human embryonic stem cell-derived 3D neural tissue model for neurotoxicity testing. Toxicol \u003cem\u003eIn Vitro\u003c/em\u003e 38: 124-135.\u003c/p\u003e\r\n\r\n\u003cp\u003eSchneider JS, Kidd SK, Anderson DW. 2013. Influence of developmental lead exposure on expression of DNA methyltransferases and methyl cytosine-binding proteins in hippocampus. Toxicol Lett 217(1): 75-81.\u003c/p\u003e\r\n\r\n\u003cp\u003eSchoemaker D, Gauthier S, Pruessner JC. 2014. Recollection and familiarity in aging individuals with mild cognitive impairment and Alzheimer\u0026#39;s disease: a literature review. Neuropsychology review 24(3): 313-331.\u003c/p\u003e\r\n\r\n\u003cp\u003eSchwartz BS, Stewart WF, Bolla KI, Simon PD, Bandeen-Roche K, Gordon PB, et al. 2000. Past adult lead exposure is associated with longitudinal decline in cognitive function. Neurology 55(8): 1144-1150.\u003c/p\u003e\r\n\r\n\u003cp\u003eSchwartz MP, Hou Z, Propson NE, Zhang J, Engstrom CJ, Santos Costa V, et al. 2015. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity. Proc Natl Acad Sci U S A 112(40): 12516-12521.\u003c/p\u003e\r\n\r\n\u003cp\u003eSherer, T.B., Betarbet, R., Greenamyre, J.T., 2002. Environment, mitochondria, and Parkinson\u0026#39;s disease. Neuroscientist 8(3), 192-197.\u003c/p\u003e\r\n\r\n\u003cp\u003eSilva AJ, Paylor R, Wehner JM, Tonegawa S. 1992. Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice. Science 257: 206-211.\u003c/p\u003e\r\n\r\n\u003cp\u003eSteece-Collier, K., Maries, E., Kordower, J.H., 2002. Etiology of Parkinson\u0026#39;s disease: Genetics and environment revisited. Proc Natl Acad Sci U S A 99(22), 13972-13974.\u003c/p\u003e\r\n\r\n\u003cp\u003eStewart WF, Schwartz BS, Davatzikos C, Shen D, Liu D, Wu X, et al. 2006. Past adult lead exposure is linked to neurodegeneration measured by brain MRI. Neurology 66(10): 1476-1484.\u003c/p\u003e\r\n\r\n\u003cp\u003eTansey MG, Goldberg MS. 2009. Neuroinflammation in Parkinson\u0026#39;s disease: Its role in neuronal death and implications for therapeutic intervention. Neurobiol Dis.\u003c/p\u003e\r\n\r\n\u003cp\u003eTartaglione, A.M., Venerosi, A., Calamandrei, G., 2016. Early-Life Toxic Insults and Onset of Sporadic Neurodegenerative Diseases-an Overview of Experimental Studies. Curr Top Behav Neurosci 29, 231-264.\u003c/p\u003e\r\n\r\n\u003cp\u003eTsang, F., Soong, T.W., 2003. Interactions between environmental and genetic factors in the pathophysiology of Parkinson\u0026#39;s disease. IUBMB Life 55(6), 323-327.\u003c/p\u003e\r\n\r\n\u003cp\u003eVerkhratsky A, Parpura V, Pekna M, Pekny M, Sofroniew M. 2014. Glia in the pathogenesis of neurodegenerative diseases. Biochemical Society Transactions 42(5): 1291-1301.\u003c/p\u003e\r\n\r\n\u003cp\u003eWalters JL, Paule MG. 2017. Review of preclinical studies on pediatric general anesthesia-induced developmental neurotoxicity. Neurotoxicol Teratol 60: 2-23.\u003c/p\u003e\r\n\r\n\u003cp\u003eWayman GA, Lee YS, Tokomitsu H, Silva A, Soderling TR. (2008) Calmodulin-kinases: modulators of neuronal development and plasticity. Neuron 59: 914-931.\u003c/p\u003e\r\n\r\n\u003cp\u003eWhitton PS. 2007. Inflammation as a causative factor in the aetiology of Parkinson\u0026#39;s disease. Br J Pharmacol 150(8): 963-976.\u003c/p\u003e\r\n\r\n\u003cp\u003eXiao Y, Fu H, Han X, Hu X, Gu H, Chen Y, et al. 2014. Role of synaptic structural plasticity in impairments of spatial learning and memory induced by developmental lead exposure in Wistar rats. PLoS One 9(12): e115556.\u003c/p\u003e\r\n\r\n\u003cp\u003eZawia NH, Basha MR. 2005. Environmental risk factors and the developmental basis for Alzheimer\u0026#39;s disease. Rev Neurosci 16(4): 325\u003c/p\u003e\r\n\r\n\u003cp\u003eZurich M-G, Eskes C, Honegger P, B\u0026eacute;rode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate \u003cem\u003ein vitro\u003c/em\u003e: Implication of glial reactions. J Neurosc Res 70: 108-116.\u003c/p\u003e\r\n\u003c/div\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003eThe aim of this AOP is to capture the KEs and KERs that occur after chronic binding of antagonist to NMDA receptors in neurons of hippocampus and cortex during brain development and that lead to neurodegeneration with impairment in learning and memory in later life. \u0026nbsp;Neurodegenreation with accumulation of amyloid plaques and hyperphosphorylated tau, as well as cognitive deficit are associated with Alzheimer-type neurodegeneration. Currently, the hypothesis of Landrigan et al., (2005) of developmental origins of neurodegenerative diseases has been demonstrated in monkeys, in rats, mice \u0026nbsp;and in zebrafish following Pb treatment (Zawia and Basha, 2005; Basha and Reddy, 2010; Bihaqi et al., 2014a; Bihaqi et al., 2014b ; Lee and Freeman, 2014). There is strong agreement that Alzheimer\u0026#39;s disease is progressive and that neurodegeneration is occuring mainly in hippocampus and cortex, associated with cognitive deficits (Schoemaker et al., 2014). This AOP uses the MIE and several KEs of the AOP 13 entitled \u0026quot;Binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities \u0026quot;, with an additional KE: neuroinflammation and two AOs: an AO at the organ level: Neurodegeneration in hippocampus and cortex and an AO at the organism level: Impairment of learning and memory. \u0026nbsp;Impairment of learning and memory is the same AO as in AOP 13, but the point is that this AO is detected when the brain is aging, and it is due to neurodegeneration with accumulation of amyloid peptides and tau hyperphosphorylation. The recent review by Tartatglione and coworkers (2016) is a very good summary of the challenges and experimental studies described in this AOP.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eDevelopmental Pb exposure has adverse effects on cognitive functioning that can persist into adulthood and may be exacerbated with aging (Schneider et al., 2013). Such delayed effects may be due to epigenetic effects of developmental Pb exposure on DNA methylation mediated at least in part through dysregulation of methyltransferases observed often at the lowest level of exposure (Schneider et al., 2013). In addition, key neurodevelopmental events, such as neural differentiation, cell migration and network formation may be modulated by Pb exposure, predisposing the brain for alterations in higher brain functions, such as learning and memory, and this at different ages (for review, see Aschner et al., 2017). The fact that neuroinflammation triggered during early brain development was shown to cause Alzheimer-like pathology when aging (Krstic et al., 2012), suggests that chronic neuroinflammation may play a causal role in cognitive decline in aging. A recent report described a mechanistic link between chronic inflammation and aging microglia; and a causal role of aging microglia in neurodegenerative cognitive deficits: A sirtuin 1 (SIRT1) deficiency was observed in aging microglia, leading to a selective activation of IL1-b transcription mediated through hypomethylation of IL-1b proximal promoter exacerbating aging or tau-associated cognitive deficits (Cho et al. 2015). Taken together, these data suggest that Pb-induced neuroinflammation during brain development may underlie the delayed effects on cognitive deficits in aging, as depicted in the proposed AOP\u003c/p\u003e\r\n","background":"","user_defined_mie":"201: Binding of antagonist, NMDA receptors","user_defined_ao":"352: N/A, Neurodegeneration and 341: Impairment, Learning and memory","oecd_project":"1.13","oecd_status_id":1,"graphical_representation_image_uid":"2016/12/12/8s0856n8ou_Diapositive1.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2018-06-13T07:48:06.000-04:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":10,"handbook_id":1,"project_129":false},{"id":13,"title":"Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities","short_name":"Binding of antagonist to NMDARs impairs cognition","corresponding_author_id":30,"abstract":"\u003cp\u003eIt is well documented and accepted that learning and memory processes rely on physiological functioning of the glutamate receptor N-methyl-D-aspartate (NMDAR). Both animal and human studies investigating NMDA itself, experiments with NMDAR antagonists and mutant mice lacking NMDAR subunits strongly support this statement (Rezvani, 2006). Activation of NMDARs results in long-term potentiation (LTP), which is related to increased synaptic strength, plasticity and memory formation in the hippocampus (Johnston et al., 2009). LTP induced by activation of NMDA receptors has been found to be elevated in the developing rodent brain compared to the mature brain, partially due to \u0026#39;developmental switch\u0026#39; of the NMDAR 2A and 2B subunits (Johnston et al., 2009). Activation of the NMDAR also enhances brain derived neurotrophic factor (BDNF) release, which promotes neuronal survival, differentiation and synaptogenesis (Tyler et al., 2002; Johnston et al., 2009). Consequently, the blockage of NMDAR by chemical substances during synaptogenesis disrupts neuronal network formation resulting in the impairment of learning and memory processes (Toscano and Guilarte, 2005). This AOP is relevant to developmental neurotoxicity (DNT). The molecular initiating event (MIE) is described as the chronic binding of antagonist to NMDAR in neurons during synaptogenesis (development) in hippocampus (one of the critical brain structures for learning and memory formation). One of the chemicals that blocks NMDAR after chronic exposure is lead (Pb\u003csup\u003e2+\u003c/sup\u003e), a well-known developmental neurotoxicant.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":1,"authors":"\u003cp\u003eMagdalini Sachana, Sharon Munn, Anna Bal-Price\u003c/p\u003e\r\n\r\n\u003cp\u003eJoint Research Centre Institute for Health and Consumer Protection Systems Toxicology Unit Via E. Fermi 2749 - 21020 - Ispra (VA) -Italy\u003c/p\u003e\r\n\r\n\u003cp\u003eCorresponding author: anna.price@ec.europa.eu\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003e\u003cstrong\u003eLife Stage Applicability:\u003c/strong\u003e This AOP is applicable only for specific period of brain development that is the time of synaptogenesis. This vulnerable period of synaptogenesis appears to happen in different developmental stages across species. For example, in rodents primarily synaptogenesis occurs during the first two weeks after birth. For rhesus monkeys, this period ranges from approximately 115-day gestation up to PND 60. In humans, it starts from the third trimester of pregnancy and continues 2-3 years following birth (Bai et al., 2013). Furthermore, synaptogenesis does not happen in a uniform way in all brain regions and there are important differences between the times of appearance of the main two types of synapses (reviewed in Erecinska et al., 2004). For example, in rat hippocampus excitatory synapses are well established or fully mature within the two first postnatal weeks, whereas inhibitory synapses cannot be found prior to PND 18, after which it increases steadily to reach adult levels at PND 28. In addition, in rat neostriatal neurons the excitatory responses to both cortical and thalamic stimuli can be observed by PND 6, but the long-lasting hyperpolarization and late depolarization is never seen before PND 12.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\r\n\u003cp\u003e\u003cstrong\u003eTaxonomic Applicability:\u003c/strong\u003e The data used to support the KERs in this AOP derives from experimental studies conducted in rats and mice or cell cultures of similar origin as well as from human epidemiological studies. The majority of the KEs in this AOP seem to be highly conserved across species. It remains to be proved if these KERs of the present AOP are also applicable for other species rather than human, primates, rats and mice.\u003c/p\u003e\r\n\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eSex Applicability:\u003c/strong\u003e The majority of the studies addressing the KEs and KERs of this AOP were carried out mainly in male laboratory animals. Few studies are available in females and some of them compare the effects between females and males. It appears that this AOP is applicable for both females and males.\u003c/p\u003e\r\n","key_event_essentiality":"\u003cp\u003e\u003cstrong\u003e1) Essentiality of the MIE: binding of antagonist to NMDAR in neurons during synaptogenesis in hippocampus and cortex\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\r\n\u003cp\u003e\u0026nbsp;\r\n\u003cp\u003eThe MIE is defined and described above as the binding of antagonist to NMDA receptor in neurons during development in hippocampus and cortex (the critical brain structures for learning and memory formation). Activation of NMDA receptors results in long-term potentiation (LTP), which is related to increased synaptic strength and memory formation in the hippocampus (Johnston et al., 2009). LTP induced by activation of NMDA receptors has been found to be elevated in the developing rodent brain compared to the mature brain, partially due to \u0026quot;developmental switch\u0026quot; of the NMDAR 2A and 2B subunits (Johnston et al., 2009).\u003c/p\u003e\r\n\u003c/p\u003e\r\n\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of MIE (binding of antagonist to NMDAR in neurons during synaptogenesis in hippocampus and cortex) for AO (Impairment of learning and memory)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: It is well documented that learning and memory processes rely on physiological functioning of NMDA receptors. The essentiality of the MIE has been demonstrated in both animal and human studies investigating NMDA itself, NMDA receptors antagonists and mutant mice lacking NMDA receptor subunits (reviewed in Haberny et al., 2002; Rezvani, 2006 and Granger et al., 2011). NMDA systemically administered in rats, has been shown to potentiate cognitive functions (Rezvani, 2006). There are various studies dealing with specific NMDA receptor subunit gene knock-out leading to a variety of phenotypes. Depending on the endogenous levels of NMDAR subunits, the pattern of their expression and their importance in developmental processes, the loss of a subunit may lead from early embryonic lethality, to mild neurobehavioral impairment up to neuronal disorders that manifest learning and memory deficits (reviewed in Rezvani, 2006 and Granger et al., 2011). Mutant mice lacking NR1 gene have shown perinatal lethality, whereas transgenic mice lacking NR1 subunit in the CA1 region of the hippocampus show both defective LTP and severe deficits in both spatial and nonspatial learning (Shimizu et al., 2000; Tsien et al., 1996). A similar impairment of LTP, long-term depression (LTD), and spatial memory has been seen with CA1-specific NR2B deletion (Brigman et al. 2010). However, LTP has been normal in postnatal forebrain knock-out of NR2A in mice, even though spatial memory has been impaired, probably because of the severe reduction observed in overall excitatory transmission (Shimshek et al., 2006), while the inactivation of the same gene has led to reduced hippocampal LTP and spatial learning (Sakimura et al., 1995). Furthermore, a NR2B transgenic (Tg) line of mice has been developed, in which the NMDA-receptor function has been increased, showing both larger LTP in the hippocampus and superior learning and memory (Tang et al., 1999). Finally, depletion of both NR2A and NR2B in single neurons has shown alteration in synaptic development (Gray et al., 2011). Interestingly, during development, especially during postnatal days (PND) 7-14 in rodents, the central nervous system (CNS) exhibits increased susceptibility to toxic insults that affect NMDA receptors (Haberny et al., 2002). This increased susceptibility has been suggested to be related to the elevated expression of specific NMDA receptor subunits (Miyamoto et al., 2001). Because of the critical role of the NMDA receptor system in brain development, the exposure to antagonists of NMDA receptors can have long-lasting and severe effects (Behar et al., 1999). NMDA-receptor antagonists such as MK-801, ketamine, phencyclidine (PCP) and 2-amino-5-phosphonopentanoate (AP5 or APV) have been extensively used to study the role of NMDA in learning and memory in developing organisms. Both acute and subchronic administration of NMDA-receptor antagonists in several laboratory animals has been shown to impair performance on tasks that seem to depend upon hippocampal functions (reviewed in Rezvani, 2006; Haberny et al., 2002). The developmental neurotoxicity of several agents, including methylmercury, lead, and ethanol is also thought to result from interaction of these substances with the NMDA receptor system (Guilarte, 1997; Guilarte and McGlothan, 1998; Ikonomidou et al., 2000; Kumari and Ticku, 1998; Miyamoto et al., 2001).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of MIE (binding of antagonist to NMDAR in neurons during synaptogenesis in hippocampus and cortex for KE (aberrant dendritic morphology)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eMODERATE\u003c/strong\u003e\u003c/em\u003e: The cortex-restricted knockout of NR1 causes refinement in dendritic arborisation in cortex and loss of patterning (Iwasato et al., 2000; Lee et al., 2005). Similar alteration in dendritic arbor has also been identified after depletion of both NR2A and NR2B subunits in isolated neurons (Espinosa et al., 2009). Blockade of NMDA receptor with APV has shown decrease of dendritic growth rate in some in vivo experimental approaches (Rajan et al., 1999; Rajan and Cline, 1998). However, other studies have reported increase in dendritic spine number and dendritic branching after chronic APV-treatment both in vivo and in vitro (Rocha and Sur, 1995; McAllister et al., 1996). This discrepancy is possibly attributed to the different developmental expression of NMDA receptor subunits that triggers distinct intracellular signaling pathways linking NMDAR function to different morphological findings.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of MIE (binding of antagonist to NMDAR in neurons during synaptogenesis in hippocampus and cortex) for KE (cell death)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: The essential role of NMDA receptors in survival during early cortical development has been pointed out both in in vitro (Hwang et al., 1999; Yoon et al., 2003) and in vivo rodent studies (Ikonomidou et al., 1999). NMDA receptor deficient mice have revealed the importance of this receptor for neuronal survival during development as an approximately 2-fold increase in developmental cell death has been observed in these transgenic mice, which was caspase-3 and Bax dependent (Adams et al., 2004; Rivero Vaccari et al., 2006).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of MIE for KE (decreased neuronal network function)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: The NMDA receptor is associated with circuit formation and function at the developmental stage of an organism as a number of antagonists of this receptor importantly disrupt the neuronal circuit (Simon et al., 1992). Hence, the nature of evidence for the essentiality of the MIE is High (Strong).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e2) Essentiality of the KE (Inhibition of NMDA receptors)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of KE (Inhibition of NMDA receptors) for AO (Impairment of learning and memory)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: The noncompetitive antagonist MK-801 has been shown to induce dose-dependent impairment of learning and memory (Wong et al., 1986) and data on rodent models has been recently reviewed in van der Staay et al. 2011. Learning impairments induced by NMDA receptor blockade using MK-801 have also been reported in nonhuman primates (Ogura and Aigner, 1993). Moreover there are human studies demonstrating that NMDA-receptor inhibition impairs learning and memory processes (reviewed in Rezvani, 2006).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e3) Essentiality of the KE (Decreased Calcium influx)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of KE (Decreased Calcium influx) for AO (Impairment of learning and memory)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: In the nervous system, many intracellular responses to modified Ca2+ levels are mediated by calcium/calmodulin-regulated protein kinases (CaMKs), a family of protein kinases that are initially modulated by binding of Ca2+ to CaM and subsequently by protein phosphorylation (Wayman et al., 2008). Multifunctional CaMKs, such as CaMKII and members of CaMK cascade (CaMKK, CaMKI and CaMKIV) are highly abundant in CNS and regulate different protein substrates (Soderling, 1999). Mice with a mutation in the alpha- CaMKII that is abundantly found in the hippocampus have shown spatial learning impairments, whereas some types of non-spatial learning peocesses have not been affected (Silva et al., 1992).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e4) Essentiality of KE (Decreased levels of BDNF)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of KE (Decreased levels of BDNF) for AO (Impairment of learning and memory)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: BDNF serves essential functions in the brain development and more specific in synaptic plasticity (Poo, 2001) and is crucial for learning and memory processes (Lu et al., 2008). The action of BDNF signaling on synapses happens within seconds of its release (Kovalchuk et al., 2004) and strengthens LTP processes, a cellular model for learning and memory, via sustained TrkB activation as a result of elevated transcription of BDNF (Kang and Schuman, 1996; Nagappan and Lu, 2005). This positive transcriptional feedback happens through TrkB-mediated CREB activation and increases gene transcription of BDNF (Lu et al., 2008). Furthermore, there are experimental evidence showing that loss of BDNF through transgenic models or pharmacological manipulation leads to impaired LTP (Patterson et al., 1996; Monteggia et al., 2004) and decreased learning and memory (Lu et al., 2008). The important role for BDNF in LTP and learning and memory is suggested from numerous studies in rodents. Hippocampal LTP is impaired in mice lacking BDNF in their neurons, and BDNF enhances LTP in the hippocampus and visual cortex (reviewed in Mattson, 2008). BDNF can also be released from neurons during LTP and possibly recycled and used for LTP maintenance. In learning and memory enhancement studies, it has been found that dietary energy restriction (which enhances synaptic plasticity) increases the production of BDNF and glial cells derived neurotrophic factor (reviewed in Mattson, 2008). In humans, a common single-nucleotide polymorphism in the Bdnf gene results in poor performance on memory tasks and may contribute to the pathogenesis of depression and anxiety disorders (reviewed in Cohen and Greenberg, 2008). Similarly, the transgenic mice with such mutation display defects in learning and memory tasks as well as anxiety-related behaviours (reviewed in Cohen and Greenberg, 2008). BDNF has also been shown to play pivotal role in a variety of learning paradigms in a variety of animal models such as mice, monkeys, zebra finches and chicks (reviewed in Tyler et al., 2002).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e5) Essentiality of KE (Cell death)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of KE (Cell death) for AO (Impairment of learning and memory)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: Several experimental studies dealing with postnatal administration of NMDA receptor antagonists such as MK-801, ketamine or ethanol have shown a devastating cell apoptotic degeneration in several brain regions of animals models, resulting in learning deficits (reviewed in Fredriksson and Archer, 2004; Creeley and Olney, 2013). The apoptosis induced in developing brain after exposure to NMDA receptor antagonists is not reversible although the developing brain has plasticity properties that may allow to a certain degree to compensate for neuronal losses. This severe bilaterally symmetrical neuronal losses in both hemispheres that occurs by treatment with NMDA receptor antagonists leads to neurobehavioral disorders including learning and memory deficits (Creeley and Olney, 2013).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e6) Essentiality of the KE (Decreased presynaptic release of glutamate)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of KE (Decreased presynaptic release of glutamate) for AO (Impairment of learning and memory)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: Riedel et al. 2003 have reviewed data available that is related to the understanding of the role of glutamate and its different receptor subtypes in learning and memory, focusing mainly in psychopharmacological in vivo studies conducted in rodents and primates. Furthermore, this review has included literature on long-term potentiation (LTP) and long-term depression (LTD), the most commonly used models for studying the cellular mechanisms underlying memory formation in relation to glutamate rather than exploring relevant mechanistic data. Classical conditioning of a tone-shock association (commonly used to study learning and memory) causes a lasting increase in glutamate release in dentate gyrus synaptosomes, whereas blockade of NMDA receptors during learning prevents conditioning and the change in glutamate release (Redini-Del Negro and Laroche, 1993). It is worth mentioning that there are two types of LTP, the NMDA receptor-dependent and the NMDA receptor-independent. The later type of LTP is induced presynaptically and strongly activates presynaptic Ca2+ channels, which results in an increase in cAMP and activation of protein kinase A that is believed to be involved in the long-lasting enhancement of glutamate release from the presynaptic terminal. This type of LTP has been observed at mossy fiber-CA3 synapses in the hippocampus or at parallel fiber-Purkinje cell synapses in the cerebellum (Manabe, 2009).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e7) Essentiality of the KE (Aberrant dendritic morphology)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of KE (Aberrant dendritic morphology) for AO (Impairment of learning and memory)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: Spine morphology is considered to be an important morphological unit for establishing learning and memory (Sekino et al., 2007). As dendrites are the postsynaptic site of most synaptic contacts, dendritic development determines the number and pattern of synapses received by each neuron (McAllistair, 2000). Defects induced in dendritic growth are often leading to severe neurodevelopmental disorders such as mental retardation (Purpura, 1975). Thus, the proper growth and arborization of dendrites are crucial for proper functioning of the nervous system. Changes in spine formation have been found to be involved in impairment of learning and memory in live animals (Yang et al. 2009; Roberts et al. 2010). Electrical activity-dependent changes in the number as well as in the size and shape of dendritic spines have been strongly related to some forms of learning (reviewed in Holtmaat and Svoboda, 2009). In mouse, motor cortex learning leads to dendritic spine remodeling associated with the degree of behavioral improvement suggesting a crucial role for structural plasticity during memory formation (Yang et al., 2009 and Fu et al., 2012). Furthermore, accumulating evidence indicates that experience-dependent plasticity of specific circuits in the somatosensory and visual cortex involves structural changes at dendritic spines (Holtmaat and Svoboda, 2009).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e8) Essentiality of the KE (Decreased synaptogenesis)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of KE (Decreased synaptogenesis) for AO (Impairment of learning and memory)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: Learning and memory result from plastic events that modify the way neurons communicate with each other (Bear, 1996). Plastic events are considered changes in the structure, distribution and number of synapses and it has been suggested that morphological events like these underlie memory formation (Rusakov et al., 1997; Woolf, 1998; Klintsova and Greenough, 1999). In mutant mice lacking PSD-95, it has been recorded increase of NMDA-dependent LTP, at different frequencies of synaptic stimulation that cause severe impaired spatial learning, without thought affecting the synaptic NMDA receptor currents, subunit expression, localization and synaptic morphology (Migaud et al., 1998). Furthermore, recent genetic screening in human subjects and neurobehavioural studies in transgenic mice have suggested that loss of synaptophysin plays important role in mental retardation and/or learning deficits (Schmitt et al., 2009; Tarpey et al., 2009).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003e9) Essentiality of the KE (Decreased neuronal network function)\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eEssentiality of KE (Decreased neuronal network function ) for AO (Impairment of learning and memory)\u003c/strong\u003e is \u003cem\u003e\u003cstrong\u003eSTRONG\u003c/strong\u003e\u003c/em\u003e: It is well understood and documented that the ability of neurons to communicate with each other is based on neural network formation that relies on functional synapse establishment (Col\u0026oacute;n-Ramos, 2009). The connectivity and functionality of neural networks depends on where and when synapses are formed. Therefore, the decreased synapse formation during the process of synaptogenesis is detrimental and leads to decrease of neural network formation and function. The neuronal electrical activity dependence on synapse formation and is critical for proper neuronal communication. Alterations in synaptic connectivity lead to refinement of neuronal networks during development (Cline and Haas, 2008). Indeed, knockdown of PSD-95 (postsynaptic protein) blocks the functional and morphological development of glutamatergic synapses (Ehrlich et al., 2007).\u003c/p\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003eThe table provides a summary of the biological plausibility and empirical support for each KER described in this AOP based on \u0026quot;Annex 1: Guidance for assessing relative level of confidence in the overall AOP based on rank ordered elements\u0026quot; found in User\u0026#39;s Handbook.\u003c/p\u003e\r\n\r\n\u003cp\u003eMore information about the evidence that support these KERs and the relevant literature can be found in each KER description.\u003c/p\u003e\r\n\r\n\u003cp\u003eThe main reason for the overall scoring is that for the majority of KERs, the KEup and KEdown have not been investigated simultaneously in the same study.\u003c/p\u003e\r\n\r\n\u003ctable\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u003cstrong\u003eKERs WoE\u003c/strong\u003e\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u003cstrong\u003eBiological plausibility\u003c/strong\u003e\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u003cstrong\u003eDoes KEup occurs at lower doses than KEdown?\u003c/strong\u003e\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u003cstrong\u003eDoes KEup occurs at earlier time points than KE down?\u003c/strong\u003e\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u003cstrong\u003eIs there higher incidence of KEup than of KEdown?\u003c/strong\u003e\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u003cstrong\u003eInconsistencies/Uncertainties\u003c/strong\u003e\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eNMDARs, Binding of antagonist Directly Leads to NMDARs, Inhibition\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eN/A\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eYes\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eN/A\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eLimited conflicting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eNMDARs, Inhibition Directly Leads to Calcium influx, Decreased\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eSame dose\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eYes\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eLimited conficting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eCalcium influx, Decreased Indirectly Leads to Release of BDNF, Reduced\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eLimited conficting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eRelease of BDNF, Reduced Indirectly Leads to Dendritic morphology, Aberrant\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eYes\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNo conflicting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eRelease of BDNF, Reduced Indirectly Leads to Cell death, N/A\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eYes\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eLimited conficting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eRelease of BDNF, Reduced Indirectly Leads to Presynaptic release of glutamate, Reduced\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eLimited conficting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eCell death, N/A Indirectly Leads to Synaptogenesis, Decreased\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eYes\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eLimited conficting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eDendritic morphology, Aberrant Indirectly Leads to Synaptogenesis, Decreased\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot always\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNo conflicting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003ePresynaptic release of glutamate, Reduced Indirectly Leads to Synaptogenesis, Decreased\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNo conflicting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eSynaptogenesis, Decreased Directly Leads to Neuronal network function, Decreased\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eExtensive understanding\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNo conflicting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003eNeuronal network function, Decreased Indirectly Leads to Learning and memory, Impairment\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eScientific understanding is not completely established\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eYes\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eNot investigated\u003c/td\u003e\r\n\t\t\t\u003ctd\u003eLimited conficting data\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n","quantitative_considerations":"\u003cp\u003eA quantitative structure activity relationship (QSAR) model has been developed based on various molecular parameters that have been calculated for a series of competitive NMDA antagonists with known activity values and these parameters have been applied to make a regression analysis which provides a model that relates the computationally calculated parameters to experimentally determined activity values (Korkut and Varnali, 2003).\u003c/p\u003e\r\n\r\n\u003cp\u003eRecently, a QSAR model for non- competitive antagonists of NMDA receptor based on a series of 48 substituted MK-801 derivatives has been established (Chtitaa et al., 2015). In this paper, a quantitative model has been proposed and there has also been an attempt to interpret the activity of the compounds relying on the multivariate statistical analyses. By this approach, they have been able to predict the inhibitory activity of a set of new designed compounds (Chtitaa et al., 2015).\u003c/p\u003e\r\n\r\n\u003cp\u003e2D- and 3D-QSAR models have also been developed to establish the structural requirements for pyrazine and related derivatives for being NR2B selective NMDA receptor antagonists (Zambre et al., 2015).\u003c/p\u003e\r\n","optional_considerations":"\u003cp\u003eExposure to xenobiotics can potentially affect the nervous system resulting in neurobehavioral alterations and/or neurological clinical symptoms. To assess the neurotoxic properties of compounds, current testing largely relies on neurobehavioural tests in laboratory animals, histopathological analysis, neurochemical and occasionally electrophysiological observations. Throughout the years, a significant number of methods have been developed to assess neurobehaviour in laboratory animals and a comprehensive summary of them can be found in OECD Series on testing and assessment, number 20, Guidance Document for Neurotoxicity Testing (2004). Learning and memory is an important endpoint and a wide variety of tests to assess chemical effects on cognitive functions is available and used for the study of neurotoxicity in adult and young laboratory animals. Some of these tests that allow the appreciation of cognitive function in laboratory animals are: habituation, ethologically based anxiety tests (elevated plus maze test, black and white box test, social interaction test), conditioned taste aversion (CTA), active avoidance, passive avoidance, spatial mazes (Morris water maze, Biel water maze, T-maze), conditional discrimination (simple discrimination, matching to sample), delayed discrimination (delayed matching-to-sample, delayed alternation) and eye-blink conditioning.\u003c/p\u003e\r\n\r\n\u003cp\u003eThe US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 and OECD 426, respectively) require testing of learning and memory. These DNT Guidelines have been used to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009). Also in the scope of the OECD Test Guideline for Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test (422) and OECD Test Guideline for Extended One-Generation Reproductive Toxicity Study (443), learning and memory testing may have potential to be applied in the context of DNT studies. These DNT guidelines are based entirely on in vivo experiments, which are costly, time consuming, and unsuitable for testing a larger number of chemicals. For these reasons, there is currently no regulatory request for DNT studies prior to registration of new chemicals and recommendations for DNT testing are only based on certain triggers such as structural similarity with known reproductive toxicants, concerns for endocrine disruption, results from other toxicity studies, and the anticipated use and human exposure patterns. At the same time the published data strongly suggest that environmental chemicals contribute to the observed increase in children neurodevelopmental disorders such as lowered IQ, learning disabilities, attention deficit hyperactivity disorder (ADHD) and, in particular, autism. This highlights the pressing need for standardised alternative methodologies that can more rapidly and cost-effectively screen large numbers of chemicals for their potential to cause cognitive deficit in children.\u003c/p\u003e\r\n\r\n\u003cp\u003eThe present AOP can encourage the development of new in vitro assays test battery and the use of these alternatives to assess NMDAR inhibitors as chemicals with potential to induce impairment of children cognitive function and at the same time reduce the use of in vivo studies. Some of the KEs presented in this AOP have already been identified as endpoints to be measured during the mapping of available in vitro and alternative DNT testing methods by EFSA (Fritsche et al., 2015). In addition, the majority of KEs in this AOP has strong essentiality to induce the AO (impairment of learning and memory) and established indirect relationship with the AO that would allow not only the development of testing methods that address these specific KEs but also the understanding of the relationship between the measured KEs and the AO. The present AOP can potentially provide the basis for development of a mechanistically informed IATA for DNT. The construction of IATA for predicting DNT effects is expected to make use of more than one AOP within an interconnected network in order to take into consideration all possible biological processes that may contribute to impairment of learning and memory in developing organisms. Through this network, common KEs can emerge that should be considered during IATA construction and that may inform also assay development.\u003c/p\u003e\r\n\r\n\u003cp\u003eResults derived from assays based on the KEs of this AOP can serve to interpret and accept results that derive from non-standard test methods. Omics data from toxicogenomic, transcriptomic, proteomic, and metabolomic studies can be interpreted in a structured way following this AOP as a guide. Finally, this AOP could provide the opportunity to group chemicals using not only chemical properties but also mechanistic information that can later inform data gap filling by read-across.\u003c/p\u003e\r\n","references":"\u003cp\u003e\u003cbr /\u003e\r\nAdams SM, Rivero Vaccari JC, Corriveau RA. (2004) Pronounced cell death in the absence of NMDA receptors in the developing somatosensory thalamus. J Neurosci. 24: 9441-9450.\u003c/p\u003e\r\n\r\n\u003cp\u003eBai X, Twaroski D, Bosnjak ZJ. (2013) Modeling anesthetic developmental neurotoxicity using human stem cells. Semin Cardiothorac Vasc Anesth. 17: 276-287.\u003c/p\u003e\r\n\r\n\u003cp\u003eBear MF. (1996) A synaptic basis for memory storage in the cerebral cortex. Proc Natl Acad Sci USA 93: 13453-13459.\u003c/p\u003e\r\n\r\n\u003cp\u003eBehar TN, Scott CA, Greene CL, Wen X, Smith SV, Maric D, Liu Q-Y, Colton CA, Barker JL. (1999) Glutamate acting at NMDA receptors stimulates embryonic cortical neuronal migration. J Neurosci. 19, 4449\u0026ndash;4461.\u003c/p\u003e\r\n\r\n\u003cp\u003eBrigman JL, Wright T, Talani G, Prasad-Mulcare S, Jinde S, Seabold GK, et al. (2010) Loss of GluN2B-containing NMDA receptors in CA1 hippocampus and cortex impairs long-term depression, reduces dendritic spine density, and disrupts learning. J Neurosci. 30:4590\u0026ndash;4600.\u003c/p\u003e\r\n\r\n\u003cp\u003eChtitaa S, Larifb M, Ghamalia M, Bouachrinec M, Lakhlifia T. (2015) DFT-based QSAR Studies of MK801 derivatives for non competitive antagonists of NMDA using electronic and topological descriptors. Journal of Taibah University for Science. 9: 143-154.\u003c/p\u003e\r\n\r\n\u003cp\u003eCohen S, Greenberg ME. (2008) Communication between the synapse and the nucleus in neuronal development, plasticity and disease. Annu Rev Cell Dev Biol. 24: 183-209.\u003c/p\u003e\r\n\r\n\u003cp\u003eColon-Ramos DA. (2009) Synapse formation in developing neural circuits. Current topics in developmental biology 87: 53-79.\u003c/p\u003e\r\n\r\n\u003cp\u003eCline H, Haas K. (2008) The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: a review of the synaptotrophic hypothesis. J Physiol 586: 1509-1517.\u003c/p\u003e\r\n\r\n\u003cp\u003eCreeley CE, Olney JW. (2013) Drug-Induced Apoptosis: Mechanism by which Alcohol and Many Other Drugs can Disrupt Brain Development. Brain Sci. 3: 1153\u0026ndash;1181.\u003c/p\u003e\r\n\r\n\u003cp\u003eEhrlich I, Klein M, Rumpel S, Malinow R. (2007) PSD-95 is required for activity-driven synapse stabilization. Proceedings of the National Academy of Sciences of the United States of America 104: 4176-4181.\u003c/p\u003e\r\n\r\n\u003cp\u003eErecinska M, Cherian S, Silver IA. (2004) Energy metabolism in mammalian brain during development. Prog Neurobiol. 73: 397-445.\u003c/p\u003e\r\n\r\n\u003cp\u003eEspinosa JS, Wheeler DG, Tsien RW, Luo L. (2009) Uncoupling dendrite growth and patterning: single-cell knockout analysis of NMDA receptor 2B. Neuron 62:205\u0026ndash;217.\u003c/p\u003e\r\n\r\n\u003cp\u003eFredriksson A, Archer T. (2004) Neurobehavioural deficits associated with apoptotic neurodegeneration and vulnerability for ADHD. Neurotox Res. 6: 435\u0026ndash;456.\u003c/p\u003e\r\n\r\n\u003cp\u003eFritsche E, Alm H, Baumann J, Geerts L, H\u0026aring;kansson H, Masjosthusmann S, Witters H. (2015) Literature review on in vitro and alternative Developmental Neurotoxicity (DNT) testing methods. EFSA supporting publication 2015:EN-778. \u003ca class=\"external free\" href=\"http://www.efsa.europa.eu/en/supporting/pub/778e\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.efsa.europa.eu/en/supporting/pub/778e\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eFu M, Yu X, Lu J, Zuo Y. (2012) Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo. Nature 483: 92-95.\u003c/p\u003e\r\n\r\n\u003cp\u003eGranger AJ, Gray JA, Lu W, Nicol RA. (2011) Genetic analysis of neuronal ionotropic glutamate receptors subunits. J Physiol. 589: 4095\u0026ndash;4101.\u003c/p\u003e\r\n\r\n\u003cp\u003eGray JA, Shi Y, Usui H, During MJ, Sakimura K, Nicoll RA. (2011) Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo. Neuron. 71:1085-101.\u003c/p\u003e\r\n\r\n\u003cp\u003eGuilarte TR. (1997) Glutamatergic system and developmental lead neurotoxicity. Neurotoxicology 18, 665\u0026ndash;672.\u003c/p\u003e\r\n\r\n\u003cp\u003eGuilarte TR, McGlothan, JL. (1998) Hippocampal NMDA receptor mRNA undergoes subunit-specific changes during developmental lead exposure. Brain Res. 790, 98\u0026ndash;107.\u003c/p\u003e\r\n\r\n\u003cp\u003eHaberny KA, Paule MG, Scallet AC, Sistare FD, Lester DS, Hanig JP, Slikker W Jr. (2002) Ontogeny of the N-methyl-D-aspartate (NMDA) receptor system and susceptibility to neurotoxicity. Toxicol Sci. 68:9-17.\u003c/p\u003e\r\n\r\n\u003cp\u003eHoltmaat A, Svoboda K. (2009) Experience-dependent structural synaptic plasticity in the mammalian brain. Nat Rev Neurosci. 10: 647-658.\u003c/p\u003e\r\n\r\n\u003cp\u003eHwang JY, Kim YH, Ahn YH, Wie MB, Koh JY. (1999) N-Methyl-D-aspartate receptor blockade induces neuronal apoptosis in cortical culture. Exp Neurol. 159: 124-130.\u003c/p\u003e\r\n\r\n\u003cp\u003eIkonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vockler, J., Dikranian, K., Tenkova, T. I., Stefovska, V., Turski, L., Olney, J. W. (1999). Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283, 70\u0026ndash;74.\u003c/p\u003e\r\n\r\n\u003cp\u003eIkonomidou, C., Bittigau, P., Ishimaru, M. J., Wozniak, D. F., Koch, C., Genz, K., Price, M. T., Stefovska, V., Horster, F., Tenkova, T., Dikranian, K., and Olney, J. W. (2000) Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 287: 1056\u0026ndash;1060.\u003c/p\u003e\r\n\r\n\u003cp\u003eIwasato T, Datwani A, Wolf AM, Nishiyama H, Taguchi Y, Tonegawa S., et al. (2000) Cortex-restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex. Nature 406: 726\u0026ndash;731.\u003c/p\u003e\r\n\r\n\u003cp\u003eJohnston MV, Ishida A, Ishida WN, Matsushita HB, Nishimura A, Tsuji M. (2009) Plasticity and injury in the developing brain. Brain Dev. 31:1-10.\u003c/p\u003e\r\n\r\n\u003cp\u003eKang H, Schuman EM. (1996) A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science 273:1402\u0026ndash;1406.\u003c/p\u003e\r\n\r\n\u003cp\u003eKlintsova AY, Greenough WT. (1999) Synaptic plasticity in cortical systems. Curr Opin Neurobiol. 9: 203-208.\u003c/p\u003e\r\n\r\n\u003cp\u003eKorkut A, Varnali T. (2003) Quantitative structure activity relationship (QSAR) of competitive N-methyl-D-aspartate (NMDA) antagonists. Mol Phys 101: 3285-3291.\u003c/p\u003e\r\n\r\n\u003cp\u003eKovalchuk Y, Holthoff K, and Konnerth A. (2004) Neurotrophin action on a rapid timescale. Curr Opin Neurobiol 14:558\u0026ndash;563.\u003c/p\u003e\r\n\r\n\u003cp\u003eKumari M, Ticku MK. (1998). Ethanol and regulation of the NMDA receptor subunits in fetal cortical neuron. J Neurochem: 70, 1467\u0026ndash;1473.\u003c/p\u003e\r\n\r\n\u003cp\u003eLee LJ, Iwasato T, Ithoara S, Erzurumlu RS. (2005). Exuberant thalamocortical axon arborization in cortex-specific NMDAR1 knockout mice. J Comp Neurol. 485: 280\u0026ndash;292.\u003c/p\u003e\r\n\r\n\u003cp\u003eLu Y, Christian K, Lu B. (2008) BDNF: a key regulator for protein synthesis dependent LTP and long-term memory? Neurobiol Learn Mem. 89: 312\u0026ndash;323.\u003c/p\u003e\r\n\r\n\u003cp\u003eMakris SL, Raffaele K, Allen S, Bowers WJ, Hass U, Alleva E, Calamandrei G, Sheets L, Amcoff P, Delrue N, Crofton KM. (2009) A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426. Environ Health Perspect. 117:17-25.\u003c/p\u003e\r\n\r\n\u003cp\u003eManabe T. (2009) LTP. Encyclopedia of neuroscience. M D. Binder, N. Hirokawa and U. Windhorst (Eds). Springer-Verlag GmbH Berlin Heidelberg. pp 2188-2190\u003c/p\u003e\r\n\r\n\u003cp\u003eMattson MP. (2008) Glutamate and neurotrophic factors in neuronal plasticity and disease. Ann N Y Acad Sci. 1144: 97-112.\u003c/p\u003e\r\n\r\n\u003cp\u003eMcAllister AK, Katz LC, Lo DC. (1996) Neurotrophin regulation of cortical dendritic growth requires activity. Neuron 17: 1057.\u003c/p\u003e\r\n\r\n\u003cp\u003eMcAllistair AK. (2000) Cellular and molecular mechanisms of dendrite growth. Cereb Cortex 10: 963-973.\u003c/p\u003e\r\n\r\n\u003cp\u003eMigaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O\u0026#39;Dell TJ, Grant SG. (1998) Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396: 433-439.\u003c/p\u003e\r\n\r\n\u003cp\u003eMiyamoto K, Nakanishi H, Moriguchi S, Fukuyama N, Eto K, Wakamiya J, Murao K, Arimura K, Osame M. (2001) Involvement of enhanced sensitivity of N-methyl-D-aspartate receptors in vulnerability of developing cortical neurons to methylmercury neurotoxicity. Brain Res. 901: 252-258.\u003c/p\u003e\r\n\r\n\u003cp\u003eMonteggia LM, Barrot M, Powell CM, Berton O, Galanis V, Gemelli T, Meuth S, Nagy A, Greene RW, Nestler EJ. (2004) Essential role of brain-derived neurotrophic factor in adult hippocampal function. Proc Natl Acad Sci USA 101: 10827-10832.\u003c/p\u003e\r\n\r\n\u003cp\u003eNagappan G, Lu B. (2005) Activity-dependent modulation of the BDNF receptor TrkB: mechanisms and implications. Trends Neurosci. 28: 464-471.\u003c/p\u003e\r\n\r\n\u003cp\u003eOECD (1996) Guideline 422 on Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.\u003c/p\u003e\r\n\r\n\u003cp\u003eOECD (2007) Guideline 426 on Developmental Neurotoxicity.\u003c/p\u003e\r\n\r\n\u003cp\u003eOECD (2011) Guideline 443 on Extended One-Generation Reproductive Toxicity Study.\u003c/p\u003e\r\n\r\n\u003cp\u003eOECD (2004) Series on testing and assessment number 20, Guidance document for neurotoxicity testing.\u003c/p\u003e\r\n\r\n\u003cp\u003eOgura H, Aigner TG. (1993) MK-801 impairs recognition memory in rhesus monkeys: comparison with cholinergic drugs. J Pharmacol Exp Ther. 266: 60-64.\u003c/p\u003e\r\n\r\n\u003cp\u003ePatterson SL, Abel T, Deuel TA, Martin KC, Rose JC, and Kandel ER. (1996) Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron 16:1137\u0026ndash;1145.\u003c/p\u003e\r\n\r\n\u003cp\u003ePoo MM. (2001) Neurotrophins as synaptic modulators. Nat Rev Neurosci. 2: 24\u0026ndash;32.\u003c/p\u003e\r\n\r\n\u003cp\u003ePurpura DP. (1975) Dendritic differentiation in human cerebral cortex: normal and aberrant developmental patterns. Adv Neurol. 12: 91\u0026ndash;134.\u003c/p\u003e\r\n\r\n\u003cp\u003eRajan I, Witte S, Cline HT. (1999) NMDAR activity stabilizes presynaptic retinotectal axons and postsynaptic optic tectal cell dendrites in vivo. J Neurobiol. 38:357.\u003c/p\u003e\r\n\r\n\u003cp\u003eRajan I, Cline HT. (1998) Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo. J Neurosci. 18: 7836.\u003c/p\u003e\r\n\r\n\u003cp\u003eRedini-Del Negro C, Laroche S. (1993) Learning-induced increase in glutamate release in the dentate gyrus is blocked by the NMDA receptor antagonist AP5. Neurosci Res Commun. 13:157-165.\u003c/p\u003e\r\n\r\n\u003cp\u003eRezvani AH. (2006) Involvement of the NMDA System in Learning and Memory. In: Levin ED, Buccafusco JJ, editors. Animal Models of Cognitive Impairment. Boca Raton (FL): CRC Press; Chapter 4. Available from: \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/books/NBK2532/\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/books/NBK2532/\u003c/a\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eRiedel G, Platt B, Micheau J. (2003) Glutamate receptor function in learning and memory. Behav Brain Res. 140: 1-47.\u003c/p\u003e\r\n\r\n\u003cp\u003eRivero Vaccari JC, Casey GP, Aleem S, Park WM, Corriveau RA. (2006) NMDA receptors promote survival in somatosensory relay nuclei by inhibiting Bax-dependent developmental cell death. Proc Natl Acad Sci USA. 103: 16971-16976.\u003c/p\u003e\r\n\r\n\u003cp\u003eRoberts TF, Tschida KA, Klein ME, Mooney R. (2010) Rapid spine stabilization and synaptic enhancement at the onset of behavioural learning. Nature 463: 948-952.\u003c/p\u003e\r\n\r\n\u003cp\u003eRocha M, Sur M. (1995) Rapid acquisition of dendritic spines by visual thalamic neurons after blockade of N-methyl-D-aspartate receptors. Proc Natl Acad Sc. USA. 92: 8026.\u003c/p\u003e\r\n\r\n\u003cp\u003eRusakov DA, Davies HA, Harrison E, Diana G, Richter-Levin G, Bliss TVP, Stewart MG. (1997) Ultrastructural synaptic correlates of spatial learning in rat hippocampus. Neuroscience 80: 69-77.\u003c/p\u003e\r\n\r\n\u003cp\u003eSakimura K, Kutsuwada T, Ito I, Manabe T, Takayama C, Kushiya E, et al. (1995) Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit. Nature 373: 151\u0026ndash;155.\u003c/p\u003e\r\n\r\n\u003cp\u003eSchmitt U, Tanimoto N, Seeliger M, Schaeffel F, Leube RE. (2009) Detection of behavioral alterations and learning deficits in mice lacking synaptophysin. Neuroscience 162: 234-243.\u003c/p\u003e\r\n\r\n\u003cp\u003eSekino Y, Kojima N, Shirao T. (2007) Role of actin cytoskeleton in dendritic spine morphogenesis. Neurochem Int. 51: 92-104.\u003c/p\u003e\r\n\r\n\u003cp\u003eShimizu E, Tang YP, Rampon C, Tsien JZ. (2000) NMDA receptor-dependent synaptic reinforcement as a crucial process for memory consolidation. Science 290: 1170\u0026ndash;1174.\u003c/p\u003e\r\n\r\n\u003cp\u003eShimshek DR, Jensen V, Celikel T, Geng Y, Schupp B, Bus T, et al. (2006) Forebrain-specific glutamate receptor B deletion impairs spatial memory but not hippocampal field long-term potentiation. J Neurosci. 26: 8428-8440.\u003c/p\u003e\r\n\r\n\u003cp\u003eSilva AJ, Paylor R, Wehner JM, Tonegawa S. (1992) Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice. Science 257: 206-211.\u003c/p\u003e\r\n\r\n\u003cp\u003eSimon DK, Prusky GT, O\u0026rsquo;Leary DD, Constantine-Paton M (1992) N-methyl-d-aspartate receptor antagonists disrupt the formation of a mammalian neural map. Proc Natl Acad Sci USA 89: 10593-10597.\u003c/p\u003e\r\n\r\n\u003cp\u003eSoderling TR. (1999) Ta Ca-calmodulin-dependent protein cascade. Trends Biochem Sci. 24: 232-236.\u003c/p\u003e\r\n\r\n\u003cp\u003eTang YP, Shimizu E, Dube GR, Rampon C, Kerchner GA, Zhuo M, Liu G, Tsien JZ. (1999) Genetic enhancement of learning and memory in mice. Nature 401: 63-69.\u003c/p\u003e\r\n\r\n\u003cp\u003eTarpey PS, Smith R, Pleasance E, Whibley A, Edkins S, Hardy C, O\u0026rsquo;Meara S, Latimer C, Dicks E, Menzies A, et al. (2009) A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation. Nat Genet. 41: 535-543.\u003c/p\u003e\r\n\r\n\u003cp\u003eToscano CD, Guilarte TR. (2005) Lead neurotoxicity: From exposure to molecular effects. Brain Res Rev. 49: 529-554.\u003c/p\u003e\r\n\r\n\u003cp\u003eTsien JZ, Huerta PT, Tonegawa S. (1996) The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell 87: 1327\u0026ndash;1338.\u003c/p\u003e\r\n\r\n\u003cp\u003eTyler WJ, Alonso M, Bramham CR, Pozzo-Miller LD. (2002) From acquisition to consolidation: on the role of brain-derived neurotrophic factor signaling in hippocampal-dependent learning. Learn Mem. 9: 224\u0026ndash;237.\u003c/p\u003e\r\n\r\n\u003cp\u003evan der Staay FJ, Rutten K, Erb C, Blokland A. (2011) Effects of the cognition impairer MK-801 on learning and memory in mice and rats. Behav Brain Res. 220: 215-229.\u003c/p\u003e\r\n\r\n\u003cp\u003eWayman GA, Lee YS, Tokomitsu H, Silva A, Soderling TR. (2008) Calmodulin-kinases: modulators of neuronal development and plasticity. Neuron 59: 914-931.\u003c/p\u003e\r\n\r\n\u003cp\u003eWong EH, Kemp JA, Priestley T, Knight AR, Woodruff GN, Iversen LL. (1986) The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc Natl Acad Sci U S A. 83: 7104-7108.\u003c/p\u003e\r\n\r\n\u003cp\u003eWoolf NJ. (1998) A structural basis for memory storage in mammals. Prog Neurobiol. 55: 59-77.\u003c/p\u003e\r\n\r\n\u003cp\u003eYang G, Pan F, Gan WB. (2009) Stably maintained dendritic spines are associated with lifelong memories. Nature 462: 920-924.\u003c/p\u003e\r\n\r\n\u003cp\u003eYang J, Siao CJ, Nagappan G, Marinic T, Jing D, McGrath K. (2009) Neuronal release of proBDNF. Nat Neurosci. 12: 113-115.\u003c/p\u003e\r\n\r\n\u003cp\u003eYoon WJ, Won SJ, Ryu BR, Gwag BJ. (2003) Blockade of ionotropic glutamate receptors produces neuronal apoptosis through the Bax- cytochrome C-caspase pathway: the causative role of Ca2+deficiency. J Neurochem. 85: 525-533.\u003c/p\u003e\r\n\r\n\u003cp\u003eZambre VP, Hambarde VA, Petkar NN, Patela CN, Sawanta SD. (2015) Structural investigations by in silico modeling for designing NR2B subunit selective NMDA receptor antagonists. RSC Adv. 5: 23922-23940.\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003eThe aim of the present AOP is to construct a linear pathway that captures the KEs and KERs that occur after binding of antagonist to NMDA receptor in neurons during development in hippocampus and cortex. All KEs of the AOP are characterised by STRONG essentiality for the AO (learning and memory impairment). Similarly, the biological plausibility in the majority of KERs is rated STRONG as there is extensive mechanistic understanding. However, the empirical support for the present KERs cannot be rated high as in most occasions the KEup and KEdowm of a KER have not been investigated simultaneously under the same experimental protocol.\u003c/p\u003e\r\n","background":"","user_defined_mie":"201: Binding of antagonist, NMDA receptors","user_defined_ao":"341: Impairment, Learning and memory","oecd_project":"1.22","oecd_status_id":1,"graphical_representation_image_uid":"2016/11/29/33b546637_Binding_of_antagonist_to_NMDARs_impairs_cognition.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2019-04-04T10:00:53.000-04:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":11,"handbook_id":1,"project_129":false},{"id":14,"title":"Glucocorticoid Receptor Activation Leading to Increased Disease Susceptibility","short_name":"Glucocorticoid Receptor, Activation","corresponding_author_id":198,"abstract":"","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2026-01-11T16:56:06.000-05:00","status_id":2,"authors":"\u003cp\u003eCarlie A. LaLone, University of Minnesota, Water Resources Center, lalone.carlie@epa.gov\u003c/p\u003e\r\n","applicability_of_the_aop":"","key_event_essentiality":"","weight_of_evidence_summary":"","quantitative_considerations":"","optional_considerations":"","references":"","overall_assessment":"","background":"","user_defined_mie":"122: Activation, Glucocorticoid Receptor","user_defined_ao":"323: Increased, Disease susceptibility","oecd_project":null,"oecd_status_id":null,"graphical_representation_image_uid":null,"saaop_status_id":null,"legacy":true,"overall_assessment_file_uid":null,"changed_at":null,"development_strategy":"","known_modulating_factors":"\u003cdiv\u003e\r\n\u003ctable class=\"table table-bordered table-fullwidth\"\u003e\r\n\t\u003cthead\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003cth\u003eModulating Factor (MF)\u003c/th\u003e\r\n\t\t\t\u003cth\u003eInfluence or Outcome\u003c/th\u003e\r\n\t\t\t\u003cth\u003eKER(s) involved\u003c/th\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/thead\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\u003c/div\u003e\r\n","assigned_license_id":12,"handbook_id":1,"project_129":true},{"id":15,"title":"Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations","short_name":"Alkylation of DNA leading to heritable mutations","corresponding_author_id":61,"abstract":"\u003cp\u003eGerm cell/heritable mutations are important regulatory endpoints for international agencies interested in protecting the health of future generations. However, germ cell mutation analysis has been hampered by a lack of efficient tools. With the publication of the OECD test guideline TG488 (rodent transgene mutation assay) and new technologies (including next generation sequencing) this field is experiencing renewed focus. Indeed, regulatory approaches to assess germ cell mutagenicity were the focus of an IWGT workshop (Yauk et al., 2013). Of particular concern is the inability to address this endpoint through high-throughput screening assays (because spermatogenesis cannot be carried out in culture), and mutagenesis is an important gap in existing high-throughput tests. The motivation for developing this AOP was to provide context for new assays in this field, identify research gaps and facilitate the development of new methods.\u003c/p\u003e\r\n\r\n\u003cp\u003eIn this AOP, a compound capable of alkylating DNA is delivered to the testes causing germ cell mutations and subsequent mutations in the offspring of the exposed parents. The AOP requires uptake of the parent compound or metabolite in spermatogonia and interaction with DNA in those cells. DNA alkylation in male pre-meiotic germ cells is the molecular initiating event. A variety of different DNA adducts are formed that are subject to DNA repair; however, at high doses the repair machinery becomes saturated or overwhelmed. The fate of remaining adducts includes: (1) attempted DNA repair by alternative DNA repair machinery, or (2) no repair. Key event (KE) 1 is insufficient or incorrect DNA repair. Lack of repair can lead to replication of adducted DNA and ensuing mutations in male pre-meiotic germ cells (KE2). Mutations that do not impair spermatogenic processes will persist in these cells and eventually be present in the mature sperm. Thus, the mutations can be transmitted to the offspring (adverse outcome \u0026ndash; inherited mutations). It is well documented that mice and other animals exposed to alkylating agents develop mutations in male pre-meiotic germ cells that are then found in sperm, resulting in the transmission of mutations to their offspring. There is a significant amount of empirical evidence supporting the AOP and the overall weight of evidence is strong. Although there are some gaps surrounding some mechanistic aspects of this AOP, the overarching AOP is widely accepted and applies broadly to any species that produces sperm.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":1,"authors":"\u003cp\u003eCarole Yauk (1)*\u003c/p\u003e\r\n\r\n\u003cp\u003eIain Lambert (2)\u003c/p\u003e\r\n\r\n\u003cp\u003eFrancesco Marchetti (1)\u003c/p\u003e\r\n\r\n\u003cp\u003eGeorge Douglas (1)\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n(1) Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON, Canada\u003c/p\u003e\r\n\r\n\u003cp\u003e(2) Dept. of Biology, Carleton University, Ottawa, ON, Canada\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eCommunicating author: carole.yauk@canada.ca\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","applicability_of_the_aop":"\u003cp\u003eThis AOP is relevant exclusively to mature males and their pre-meiotic germ cells. Although not considered in this AOP, progenitor germ cells from earlier life stages may also be susceptible to induced mutations from alkylating agents, which could then be transmitted to offspring after sexual maturity. Relevant endpoints have been characterized across different taxa: (1) alkyl adduct levels in this AOP were from hamsters, mice and rats; (2) repair of alkylated DNA has been studied in prokaryotes to higher eukaryotes, including human cells in culture (while there are differences across taxa, all species have some DNA repair systems in place and it is common to extrapolate conclusions across eukaryotic species); (3) mutations in male germ cells were measured in mice and fish; and (4) mutations in offspring were measured in Drosophila, Japanese Medaka and mice. Quite generally, the AOP applies to any species that produces sperm. The similarity in spermatogenesis and in DNA repair of alkyl adducts is well documented across rodents and humans (Adler 1996). Heritable mutations are the basis of evolution and occur in every species. That mutations in sperm are transmitted to offspring in humans is best demonstrated by studies exploring the effects of ageing. Significant increases are observed in the amount of DNA damage and mutation as human males age (reviewed in Paul and Robaire 2013). Similarly, increased incidence of single nucleotide mutations and microsatellite mutation in the offspring of ageing fathers has recently been measured by advanced genomics technologies (Kong et al. 2012; Sun et al. 2012). Lifestyle factors including smoking and lower income brackets in human fathers in associated with increased minisatellite mutations in their offspring (LinSchooten et al., 2013).\u003c/p\u003e\r\n","key_event_essentiality":"\u003cp\u003eEssentiality was not directly tested for all of the KEs. The MIE cannot be \u0026lsquo;blocked\u0026rsquo; in any way to our knowledge (e.g., as you might block a receptor-binding MIE). However, as described in the KERs, enhanced DNA repair of alkylated DNA reduces mutation frequencies and reduction in repair increases mutation frequencies, supporting the essentiality of KE1 (i.e., moderate support). Correct repair of the alkylated DNA (i.e., a block of KE1) will not lead to mutation. For example, MGMT overexpression protects mgt1 mutant yeast against alkylation-induced mutation (Xiao and Fontanie 1995). In addition, Big Blue\u0026reg; mice over-expressing human AGT exhibit greatly reduced O6-methylguanine-mediated lacI and K-ras mutations in the thymus following treatment with MNU (Allay et al. 1999) relative to wild type Big Blue\u0026reg; mice. Insufficient DNA repair is well-established to lead to mutations. In addition, inactivation of MGMT sensitizes cells to alkylation-induced mutagenesis resulting in an increased number of mutations per adduct (Thomas et al. 2013).\u003c/p\u003e\r\n\r\n\u003cp\u003eThe remainder of the AOP requires transmission of mutations in sperm to offspring. There are no means to study the essentiality of mutations in sperm. Once mutations occur in male pre-meiotic germ cells, they cannot be removed to observe whether occurrence in offspring is decreased. In addition, mutations that occur in stem cells are propagated clonally and can become fixed in the spermatogonial cell population. Thus, waiting a longer period of time, or removing the exposure, is not effective in causing a decline in the mutation frequency. Therefore, the essentiality of this KE is inferred by the biology of the pathway and cannot be addressed directly with experimental evidence.\u003c/p\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003eBiological plausibility of the KERs: Strong. There is extensive understanding of the ability of alkylating agents to cause DNA adducts, the requirement for overcoming DNA repair, and the resulting mutations that arise in both somatic and germ cells. It is established that exposure to alkylating agents produced mutations in germ cells \u0026ndash; ENU is used in genetic screening to produce mutations to derive new phenotypes for research.\u003c/p\u003e\r\n\r\n\u003cp\u003eEmpirical support for the KERs: Across the KERs the degree of support ranges from weak to strong (\u003ca href=\"/wiki/index.php/File:AssessmentSummaryAop-15.pdf\" title=\"File:AssessmentSummaryAop-15.pdf\"\u003eFile:AssessmentSummaryAop-15.pdf\u003c/a\u003e - Table II). Support from somatic cells in culture contributes to moderate calls for the relationships between adduct formation, insufficient DNA repair and mutation. The weak call is based on lack of empirical data to support that mutations in germ cells are transmitted to offspring. However, increased mutation frequencies in germ cells occur following exposure to the same types of chemicals that cause increased mutations in the offspring. It should be noted that biological plausibility for this KER is strong as it is based on understanding of molecular biology and evolution. The strongest support is associated with the indirect KER linking alkylation of DNA to mutation in germ cells (KER4). This is primarily based on extensive evidence in both somatic and germ cells demonstrating that chemicals that alkylate DNA cause mutations, that alkyl adducts occur at a greater incidence than mutations at matching doses, and that alkyl adducts precede mutations. In somatic cells, work has been done on many different chemicals, whereas the germ cell data were primarily for the chemical ENU (but data were also available for a few select other chemicals) (\u003ca href=\"/wiki/index.php/File:AssessmentSummaryAop-15.pdf\" title=\"File:AssessmentSummaryAop-15.pdf\"\u003eFile:AssessmentSummaryAop-15.pdf\u003c/a\u003e - Table I, Figure 2). In addition, data are available for multiple species to support this indirect KER. There is a large degree of consistency in the germ cell literature to show that a variety of O-alkylating agents cause male germ cell mutations in many species (Drosophila, fish and rodent) and that these effects occur at many mutational loci (e.g., mutations in genes that are inherited measured with the Specific Locus Test, sperm mutations in tandem repeat DNA sequences, tandem repeat mutations in offspring, transgene mutations in sperm). Many alkylating agents have been tested to show that they create adducts in male rodent germ cells (e.g., DEN, ENU, EMS, DES), mutations in male mouse germ cells (ENU, IPMS and MNU) and mutations in the offspring of exposed male mice (ENU, MNU and IPMS). In summary, we consider the overall empirical data supporting the AOP to be MODERATE (the median call). Rank order (provided in the overall assessment Table - \u003ca href=\"/wiki/index.php/File:AssessmentSummaryAop-15.pdf\" title=\"File:AssessmentSummaryAop-15.pdf\"\u003eFile:AssessmentSummaryAop-15.pdf\u003c/a\u003e):\u003c/p\u003e\r\n\r\n\u003cp\u003eRank order of the KERs and the weight of evidence for the essentiality all point to the overall weight of evidence for this AOP as strong. Biological plausibility is strong for all KERs, with primarily moderate evidence for KER linkages and relatively few uncertainties or inconsistencies.\u003c/p\u003e\r\n","quantitative_considerations":"\u003cp\u003eAs described above, it is established that alkyl adducts, mutations in spermatogonia and mutations in offspring all increase with dose in a manner that is consistent with the AOP. Alkylation must exceed a threshold (determined by saturation of the relevant DNA repair pathways) before alkyl DNA lesions persist, and mutations subsequently begin to occur. However, the precise quantitative relationship has not been modeled. Existing data published in the literature could be mined to do this and thresholds for specific adduct types (i.e., estimates of how many adducts are needed to cause a mutation in a gene on average) have been published for certain cell types, which should theoretically correlate with germ cell mutagenicity for ENU and other alkylating agents.\u003c/p\u003e\r\n\r\n\u003cp\u003eThe quantitative relationship between mutations in sperm and mutations in the offspring has not been determined and will be locus- and mutation-type specific (e.g., stronger selection against coding mutations than non-coding mutations, which will influence transmission probability); however, although many mutations will lead to embryonic loss, a large subset of mutations is expected to be heritable and viable. It is expected that quantitative understanding of this relationship will increase as advanced single cell sequencing technologies are more developed to query mutations in sperm versus offspring. For non-coding sites (e.g., transgenic reporter genes and non-coding DNA like tandem repeats), the relationship is expected to approach 1:1.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nOverall, the variables that could be used to predict whether a heritable mutation is probable following exposure to an alkylating agent are the number and types of adducts per nucleotide (and knowledge of their repair efficiency). Generally, the probability of a mutation occurring is highly dependent on the type of adduct formed (mutagenicity of the adduct is based on repair efficiency and probability of error-free replication over the lesion) and abundance of the adducts, and could be modeled using existing published data.\u003c/p\u003e\r\n","optional_considerations":"\u003cp\u003eThe information provided in this AOP will provide context for understanding how to interpret new data produced from the rodent transgene mutation assay applied to sperm (OECD TG 488) [OECD 2013], which is being increasingly applied, as well as data produced using tandem repeat mutation assays. In addition, it is envisioned that next generation sequencing technologies will enable the analysis of germ cell mutations in human populations and the eventual discovery of human germ cell mutagens. It is important to note that the regulation of chemicals that can induce heritable effects has, to date, been based heavily on extrapolation from somatic cell data. Although regulatory agencies around the world have policies in place for germ cell mutagens, risk management based on an agent that is classified as a germ cell mutagen has not yet occurred because of lack of solid evidence that these exist. This AOP demonstrates strong evidence to support the existence of male rodent germ cell mutagens, supported by data in other species (fish, flies, birds), and strongly implies that such mutagens will also affect human germ cells.\u003c/p\u003e\r\n","references":"\u003cp\u003eAllay, E., M. Veigl and S.L. Gerson (1999), \u0026quot;Mice over-expressing human O6 alkylguanine-DNA alkyltransferase selectively reduce O6 methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea\u0026quot;, \u003cem\u003eOncogene\u003c/em\u003e, 18(25): 3783-3787.\u003c/p\u003e\r\n\r\n\u003cp\u003eBernstein, B.E., E. Birney, I. Dunham, E.D. Green, C. Gunter and M. Snyder (2012), \u0026quot;An integrated encyclopedia of DNA elements in the human genome\u0026quot;, \u003cem\u003eNature\u003c/em\u003e, 489(7414): 57-74.\u003c/p\u003e\r\n\r\n\u003cp\u003eBoyko, A.R., S.H. Williamson, A.R. Indap, J.D. Degenhardt, R.D. Hernandez, K.E. Lohmueller, M.D. Adams, S. Schmidt, J.J. Sninsky, S.R. Sunyaev, T.J. White, R. Nielsen, A.G. Clark and C.D. Bustamante (2008), \u0026quot;Assessing the evolutionary impact of amino acid mutations in the human genome\u0026quot;, \u003cem\u003ePLoS Genetics\u003c/em\u003e, 4: e1000083.\u003c/p\u003e\r\n\r\n\u003cp\u003eConrad, D.F., J.E. Keebler, M.A. DePristo, S.J. Lindsay, Y. Zhang, F. Casals, Y. Idaghdour, C.L. Hartl, C. Torroja, K.V. Garimella, M. Zilversmit, R. Cartwright, G.A. Rouleau, M. Daly, E.A. Stone, M.E. Hurles and P. Awadalla (2011) \u0026quot;Variation in genome-wide mutation rates within and between human families\u0026quot;, \u003cem\u003eNature Genetics\u003c/em\u003e, 43(7): 712-714.\u003c/p\u003e\r\n\r\n\u003cp\u003eGirirajan, S., C.D. Campbell and E.E. Eichler (2010) \u0026quot;Human Copy Number Variation and Complex Genetic Disease\u0026quot;, \u003cem\u003eAnnual Review of Genetics\u003c/em\u003e, 45: 203-226.\u003c/p\u003e\r\n\r\n\u003cp\u003eHoischen, A., B.W. van Bon, C. Gilissen, P. Arts, B. van Lier, M. Steehouwer, P. de Vries, R. de Reuver, N. Wieskamp, G. Mortier, K. Devriendt, M.A. Amorim, N. Revencu, A. Kidd, M. Barbosa, A. Turner, J. Smith, C. Oley, A. Henderson, I.M. Hayes, E.M. Thompson, H.G. Brunner, B.B. de Vries and J.A. Veltman (2010) \u0026quot;De novo mutations of SETBP1 cause Schinzel-Giedion syndrome\u0026quot;, \u003cem\u003eNat. Genet.\u003c/em\u003e, 42(6): 483-485.\u003c/p\u003e\r\n\r\n\u003cp\u003eKong, A., M.L. Frigge, G. Masson, S. Besenbacher, P. Sulem, G. Magnusson, S.A. Gudjonsson, A. Sigurdsson, A. Jonasdottir, W.S. Wong, G. Sigurdsson, G.B. Walters, S. Steinberg, H. Helgason, G. Thorleifsson, D.F. Gudbjartsson, A. Helgason, O.T. Magnusson, U. Thorsteinsdottir and K. Stefansson (2012), \u0026quot;Rate of de novo mutations and the importance of father\u0026#39;s age to disease risk\u0026quot;, \u003cem\u003eNature\u003c/em\u003e, 488(7412): 471-475.\u003c/p\u003e\r\n\r\n\u003cp\u003eKryukov, G.V., L.A. Pennacchio and S.R. Sunyaev (2007), \u0026quot;Most rare missense alleles are deleterious in humans: implications for complex disease and association studies\u0026quot;, \u003cem\u003eAmerican Journal of Human Genetics\u003c/em\u003e, 80(4): 727-739.\u003c/p\u003e\r\n\r\n\u003cp\u003eKu, C.S., V. Vasiliou and D.N. Cooper (2012), \u0026quot;A new era in the discovery of de novo mutations underlying human genetic disease\u0026quot;, \u003cem\u003eHuman Genomics\u003c/em\u003e, 12(6):27.\u003c/p\u003e\r\n\r\n\u003cp\u003eLupski, J.R. (2010), \u0026quot;New mutations and intellectual function\u0026quot;, \u003cem\u003eNature Genetics\u003c/em\u003e, 42(12): 1036-1038.\u003c/p\u003e\r\n\r\n\u003cp\u003eLinschooten, J.O., N. Verhofstad, K. Gutzkow, A.K. Olsen, C. Yauk, Y. Oligschl\u0026auml;ger, G. Brunborg, F.J. van Schooten and R.W. Godschalk (2013), \u0026ldquo;Paternal lifestyle as a potential source of germline mutations transmitted to offspring\u0026rdquo;, \u003cem\u003eFASEB J\u003c/em\u003e, 27: 2873-28749. Paul C, Robaire B. 2013. Ageing of the male germ line. Nat Rev Urol 10(4):227-234.\u003c/p\u003e\r\n\r\n\u003cp\u003eMcLaughlin, H.M., R. Sakaguchi, C. Liu, T. Igarashi, D. Pehlivan, K. Chu, R. Iyer, P. Cruz, P.F. Cherukuri, N.F. Hansen, J.C. Mullikin, Program NCS, L.G. Biesecker, T.E. Wilson, V. Ionasescu, G. Nicholson, C. Searby, K. Talbot, J.M. Vance, S. Zuchner, K. Szigeti, J.R. Lupski, Y.M. Hou, E.D. Green and A. Antonellis (2010), \u0026quot;Compound heterozygosity for loss-of-function lysyl-tRNA synthetase mutations in a patient with peripheral neuropathy\u0026quot;, \u003cem\u003eAm. J. Hum. Genet.\u003c/em\u003e, 87(4): 560-566.\u003c/p\u003e\r\n\r\n\u003cp\u003eMorrow, E.M. (2010), \u0026quot;Genomic copy number variation in disorders of cognitive development\u0026quot;, \u003cem\u003eJournal of the American Academy of Child and Adolescent Psychiatry\u003c/em\u003e, 49(11): 1091-1104.\u003c/p\u003e\r\n\r\n\u003cp\u003eO\u0026#39;Roak, B.J., L. Vives, S. Girirajan, E. Karakoc, N. Krumm, B.P. Coe, R. Levy, A. Ko, C. Lee, J.D. Smith, E.H. Turner, I.B. Stanaway, B. Vernot, M. Malig, C. Baker, B. Reilly, J.M. Akey, E. Borenstein, M.J. Rieder, D.A. Nickerson, R. Bernier, J. Shendure and E.E. Eichler (2012), \u0026quot;Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations\u0026quot;, \u003cem\u003eNature\u003c/em\u003e, 485(7397): 246-250.\u003c/p\u003e\r\n\r\n\u003cp\u003eRoach, J.C., G. Glusman, A.F. Smit, C.D. Huff, R. Hubley, P.T. Shannon, L. Rowen, K.P. Pant, N. Goodman, M. Bamshad, J. Shendure, R. Drmanac, L.B. Jorde, L. Hood, D.J. Galas (2010), \u0026quot;Analysis of genetic inheritance in a family quartet by whole-genome sequencing\u0026quot;, \u003cem\u003eScience\u003c/em\u003e, 328(5978): 636-639.\u003c/p\u003e\r\n\r\n\u003cp\u003eSun, J.X., A. Helgason, G. Masson, S.S. Ebenesersdottir, H. Li, S. Mallick, S. Gnerre, N. Patterson, A. Kong, D. Reich and K. Stefansson (2012), \u0026quot;A direct characterization of human mutation based on microsatellites\u0026quot;, \u003cem\u003eNature Genetics\u003c/em\u003e, 44(10): 1161-1165.\u003c/p\u003e\r\n\r\n\u003cp\u003eThomas, A.D., G.J. Jenkins, B. Kaina, O.G. Bodger, K.H. Tomaszowski, P.D. Lewis, S.H. Doak, G.E. Johnson (2013), \u0026quot;Influence of DNA repair on nonlinear dose-responses for mutation\u0026quot;, \u003cem\u003eToxicol. Sci.\u003c/em\u003e, 132(1): 87-95.\u003c/p\u003e\r\n\r\n\u003cp\u003eVissers, L.E., J. de Ligt, C. Gilissen, I. Janssen, M. Steehouwer, P. de Vries, B. van Lier, P. Arts, N. Wieskamp, M. del Rosario, B.W. van Bon, A. Hoischen, B.B. de Vries, H.G. Brunner, J.A. Veltman (2010), \u0026quot;A de novo paradigm for mental retardation\u0026quot;, \u003cem\u003eNature Genetics\u003c/em\u003e, 42(12): 1109-1112.\u003c/p\u003e\r\n\r\n\u003cp\u003eYauk C.L., Aardema, M.J., Benthem, J., Bishop, J.B., Dearfield, K.L., DeMarini, D.M., Dubrova, Y.E., Honma, M., Lupski, J.R., Marchetti, F., Meistrich, M.L., Pacchierotti, F., Stewart, J., Waters, M.D., Douglas, G.R. (2013), \u0026quot;Approaches for identifying germ cell mutagens: Report of the 2013 IWGT workshop on germ cell assays\u0026quot;, \u0026quot;Mutation Research Genetic Toxicolology and Environmental Mutagenesis\u0026quot;, 783: 36-54.\u003c/p\u003e\r\n\r\n\u003cp\u003eXiao, W. and T. Fontanie (1995), \u0026quot;Expression of the human MGMT O6-methylguanine DNA methyltransferase gene in a yeast alkylation-sensitive mutant: its effects on both exogenous and endogenous DNA alkylation damage\u0026quot;, \u003cem\u003eMutat. Res\u003c/em\u003e, 336(2): 133-42.\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003eBefore developing this AOP a review of the literature was undertaken to identify studies in which male germ cells were exposed to alkylating agents and measures of DNA adducts, DNA repair and mutations, as well as mutations in offspring, were evaluated. The focus of this AOP (as described in the KERs) is on O-alkylating agents, which are signficantly more mutagenic than N-alkylation chemicals. Studies where sufficient information relating to the chemicals used, dose, tissue, time-point, animal model, experimental procedures and experimental results were available were considered to assess empirical data in germ cells for each of the KEs and KERs in the AOP. The germ cell database on which the AOP was based is found in Supplementary Table I (\u003ca href=\"https://aopwiki.org/system/dragonfly/production/2017/05/19/1qoq9ky7zb_AOP15_supporting_evidence.pdf\" title=\"File:SupplementalTablesAop-15.pdf\"\u003eSupplementalTablesFigures\u003c/a\u003e) and is comprised of 32 studies. No study measured multiple KEs within it; however, for each KE there were at least two dose-response and time-series analyses for at least one alkylating agent. We consider this overall number of high quality studies to be fairly extensive evidence of the ability of O-alkylating agents to cause adducts and mutations in germ cells, and mutations in offspring, although no studies were ideally suited to establish the empirical linkages between the KERs. We thus compared results across studies where possible to attempt to do this. All of the studies either used ENU as the primary study compound, or applied ENU as one of the positive controls to assess other alkylating agents. Strong dose-response data for mutations occurring in exposed pre-meiotic germ cells and mutations in offspring are only available for ENU. The other alkylating agents show varying degrees mutagenicity, but single doses were used in most studies. Thus, the evaluation of concordance of the dose-response could only be undertaken with ENU for in vivo germ cell and heritable effects. However, where possible we used information from research on somatic cells to provide additional support for the KERs. In particular, experiments in somatic cells were necessary to assess the involvement of DNA repair in removing adducts and preventing mutations. Overall, we note that the rationale for claiming high confidence in this AOP and its KERs is based primarily on the more influential Bradford Hill consideration of biological plausibility, with decades of research having been done in somatic and germ cells on DNA damage, repair and mutation. Much of the data, then, supporting AOP evaluation derives from historical studies from the 1990\u0026rsquo;s, with less recent evidence. As noted, a primary motivation for developing this AOP was the recent release of TG 488, and newly available whole generation sequencing methods, which we expect to be increasingly applied. Thus, additional well-designed experiments that dissect the relationships between alkyl adducts, mutations in sperm, and mutations in offspring to assess essentiality and empirical support are expected in the future through application of these improved approaches. Below we describe each KE and KER in detail, using the wiki entries as a guide to the order of presentation and the content described.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","background":"\u003cp\u003eDe novo germ cell mutations are changes in the DNA sequence of sperm or egg that can be inherited by offspring. De novo mutations contribute to a wide range of human disorders including cancer, infertility, autism, schizophrenia, intellectual disability, and epilepsy (Girirajan et al. 2010; Hoischen et al. 2010; Ku et al. 2012; Lupski 2010; Morrow 2010; Vissers et al. 2010). Each child inherits, on average, approximately one de novo mutation per 100 million nucleotides delivered via the parental egg and sperm (Conrad et al. 2011; Kong et al. 2012; O\u0026#39;Roak et al. 2012; Roach et al. 2010). The precise locations and types of mutations in the genomic DNA sequence govern the outcome of these mutations (e.g., protein coding versus intergenic sequences, conserved versus non-conserved mutations, etc.). Although a large portion of human DNA is of unknown function, recent literature suggests that at least 80% of the genome is transcribed, and most DNA is expected to have a biological function (Bernstein et al. 2012). It has been estimated that the proportion of coding and splice-site base substitutions that result in truncating mutations is ~5% (Kryukov et al. 2007), and that as many as 30% of missense mutations are also likely to be highly deleterious due to loss of function (Boyko et al. 2008). When they occur in functional sites, de novo mutations can cause embryonic or fetal lethality, or if viable, can produce a broad spectrum of inherited genetic disorders. Recent estimates suggest that a human genome contains approximately 100 loss-of-function variants, with as many as 20 exhibiting complete loss of gene function (McLaughlin et al. 2010). Therefore, de novo mutations contribute to the overall population genetic disease burden. The present AOP focuses on \u003ca href=\"/wiki/index.php/DNA_alkylation_in_spermatogonia\" title=\"DNA alkylation in spermatogonia\"\u003eDNA alkylation in spermatogonia\u003c/a\u003e that causes inherited mutation transmitted via sperm, arguably one of the most well characterized modes of action in genetic toxicology. Humans are exposed to alkylating agents from external (e.g., abiotic plant materials, tobacco smoke, combustion products, chemotherapeutic agents) and internal (e.g., byproducts of oxidative damage and cellular methyl donors) sources.\u003c/p\u003e\r\n","user_defined_mie":"97: Alkylation, DNA","user_defined_ao":"336: Increase, Heritable mutations in offspring","oecd_project":"1.11","oecd_status_id":1,"graphical_representation_image_uid":"2020/04/07/7nmrf2zs6j_Inherited_Mutations.JPG","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":"2017/05/19/7s1ibrunwt_RevisedAssessmentSummaryAop_15.pdf","changed_at":null,"development_strategy":null,"known_modulating_factors":null,"assigned_license_id":13,"handbook_id":1,"project_129":false},{"id":16,"title":"Acetylcholinesterase inhibition leading to acute mortality","short_name":"AChE inhibition - acute mortality","corresponding_author_id":313,"abstract":"\u003cp\u003eThe contents of this AOP page represent an interconnected network of AOPs linking the MIE of acetylcholinesterase inhibition to the AO of acute mortality. Both AOPs include the KE of acetylcholine accumulation at the synapses, which results in excessive signaling from cholinergic neurons on a broad range of tissues throughout the body. Respiratory failure is the predominant mechanism leading to acute mortality in humans (Satoh, 2006). While these two AOPs are represented on the basis of their most plausible linkages to acute mortality, other known symptoms of acetylcholinesterase inhibition mediated through actions on other receptors and tissues may also play a role (see Russom et al. 2014). Overall, there is strong evidence supporting the linkage of acetylcholinesterase inhibition and acetylcholine accumulation with acute mortality, but the precise contribution of the different organ-level effects across different species isn\u0026rsquo;t completely understood. This network of AOPs as a whole, including the indirect KERs depicted, supports the potential utility of in vitro or short-term in vivo measures of acetylcholinesterase inhibition for identifying chemicals with potential to cause acute mortality across a broad range of species. Caution is needed when interpreting the in vitro results, however, because well known chemical initiators of these AOPs are known to require metabolic activation, which can result in false negatives. In contrast, detoxification of these compounds is sometimes deficient in the young resulting in life stage differences in response to different chemicals that act through this mechanism. Toxicokinetics is also variable across species making this a major determinant of species sensitivity. At present, quantitative understanding is not sufficiently complete to accurately predict apical outcomes or potency from in vitro measurements alone, and the chemical-specific ADME and toxicokinetic considerations will be strong determinant of quantitative outcomes.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":6,"authors":"\u003cp\u003eCHRISTINE L. RUSSOM (1), DANIEL L. VILLENEUVE* (2), VIRGINIA HENCH (3), CATAIA IVES (3), VIRGINIA C. MOSER (1), CARLIE A. LALONE (2), STEPHEN EDWARDS (3), KRISTIE SULLIVAN (4), and GERALD T. ANKLEY (2)\u003c/p\u003e\r\n\r\n\u003cp\u003e(1) U.S.\u0026nbsp;Environmental Protection Agency (Retired)\u003c/p\u003e\r\n\r\n\u003cp\u003e(2) National Health and Environmental Effects Research Laboratory, Office of Research and Development, Mid-Continent Ecology Division, US Environmental Protection Agency, Duluth, Minnesota, USA\u003c/p\u003e\r\n\r\n\u003cp\u003e(3) Research Computing Division, RTI International, Research Triangle Park, North Carolina, USA\u003c/p\u003e\r\n\r\n\u003cp\u003e(4)\u0026nbsp;Physicians Committee for Responsible Medicine,\u0026nbsp;Washington, DC, USA\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eCorresponding author for wiki entry (Villeneuve.dan@epa.gov)\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","applicability_of_the_aop":"\u003ch5 dir=\"ltr\"\u003eLife Stage Applicability\u003c/h5\u003e\r\n\r\n\u003cp dir=\"ltr\"\u003eThe key molecular target is the AChE enzyme, which appears to be available in all life stages of vertebrate and invertebrate species, although studies have found that AChE activity increases as the organism develops.\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cul\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eAChE can be inhibited by stressors from early in development throughout aging.\u0026nbsp; AChE inhibition has been measured in rat fetuses when the dam was dosed with chlorpyrifos (Lassiter et al., 1999), and also in aged rats up to 2 years of age treated with either carbaryl or methomyl (Moser et al., 2015).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eIn many species, sensitivity to stressors is greater in the young.\u0026nbsp; There are several factors that influence these age-related differences.\u0026nbsp; Intake from food and water is higher on a body weight basis, and in children, certain behaviors (crawling, hand-to-mouth) can also increase intake.\u0026nbsp; More importantly, considerable evidence shows that immature detoxification in the young account for much of the age differences. AChE inhibitors are metabolized or detoxified through a number of pathways, including hydrolysis by or binding to various esterases, and microsomal metabolism.\u0026nbsp; This leads to life-stage differences that are highly dependent on the stressor and the individual kinetic profile of each (Moser, 2011)\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eAChE in developing precocial birds were found to level off at the embryo life stage, while in altricial species the AChE activity increased as the bird developed until it reached a steady state at adulthood (Grue et al., 1981).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eA study examining the acetylcholine and cholinesterase levels in developing brains of Sprague-Dawley rats found that acetylcholine synthesis was significantly lower in neonate and juveniles when compared to the adult levels, and the neonate cholinesterase levels were significantly lower than adult levels of activity (Karanth and Pope 2003). When exposed to either parathion or chlorpyrifos, the researchers found differences in peak inhibition of cholinesterase with neonates seeing the greatest impact early in the exposure (4-24 hrs), while juveniles and adults saw the most severe impacts at 96 hrs of exposure (Karanth and Pope 2003). Exposure to pesticides did not seem to impact acetylcholine production in the rats regardless of life stage, but researchers thought the pesticides may be altering other biochemical process (e.g., choline acetyltransferase) which might ultimately impact the measures of acetylcholine (Karanth and Pope 2003).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eIn Drosophila, changes in AChE activity in the developing organism were observed, with egg stages displaying the lowest activity, and maximum activity at the pupae life stage (Parkash and Kaur 1982).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eIn mammals and birds, studies have determined that skeletal muscles of immature birds and mammals contain both butyrylcholinesterase and AChE, with butyrylcholinesterase decreasing and AChE increasing as the animal develops (Tsim et al. 1988; Berman et al, 1987).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eAnother study found that changes in AChE within the developing pig brain were dependent on the area of the brain and life stage of the animal, with significant decreases in activity within the pons and hippocampus from birth to 36 months, and no significant change in activity in the cerebellum, where activity increased up to four months of age, leveling off thereafter (Adejumo and Egbunike, 2004).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eEvidence exists that immature life stages in mammals and birds may be more sensitive to OP pesticides (see Grue et al., 1997; Grue et al., 1983; Grue et.al; 1981), although this may be related to the amount of pesticide ingested in relation to body size (Ludke et al, 1975).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eResearchers reported that frog (Bufo arenarum Hensel) embryos were more tolerant to parathion exposure than frog larvae, and associated this with the ability of the embryo AChE to recover to baseline levels faster than the larval life stage (Anguiano et al, 1994).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\u003c/ul\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003ch5 dir=\"ltr\"\u003eTaxonomic Applicability\u003c/h5\u003e\r\n\r\n\u003cp dir=\"ltr\"\u003eAlthough AChE enzyme is found in all vertebrate and invertebrate species, the activity within taxa varies.\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\r\n\t\u003cul\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eAn examination of the STRING (V9.0\u003ca href=\"http://string-db.org\"\u003e http://string-db.org\u003c/a\u003e), which is a database of known and predicted protein interactions, finds that the AChE enzyme is well conserved across vertebrate and invertebrate species.\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eA pattern of species sensitivity for OPs using terrestrial species found birds to be highly sensitive, mammals moderately sensitive, and fish and amphibians of lower sensitivity (Wallace 1992).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eTaxonomic differences as they relate to key events within the AOP may be due to a number of factors. For instance, the organism\u0026rsquo;s habitat (e.g., sediment, water, and grass), diet, body size, behavior, mobility, and skin/exoskeleton permeability could all affect the level of exposure and the ability of the chemical to reach the molecular target. A complicating factor when identifying relative species sensitivities are differences in routes of exposure which may impact the time it takes for the substance to reach the molecular target, and impacts to adsorption, distribution, metabolism, and elimination (ADME) within the organism.\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003ePhase I metabolism of phosphorothioates and phosphorodithioates by mixed function oxidases results in the more toxic oxon, which is the form that binds to AChE. Differences in species sensitivity may be impacted by the rate at which this reaction occurs, as would any mechanism related to detoxification of the oxon form. For instance, in mammals the oxon form undergoes an ester detoxification pathway which is not present in insect species, resulting in insects having a higher susceptibility to OPs than mammals (Ecobichon 2001). Similarly for procarbamates, invertebrates are able to convert the substance to the active form, while vertebrate species lack this metabolic mechanism (Stenersen 2004).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eStudies have provided evidence that differences in the AChE enzyme across taxa may better explain differences in species sensitivity. Evidence suggests that the relative activity of AChE is linked to the hydrophobic and electronic configuration of the enzyme, which directly impacts the speed and/or efficiency that a substance can bind to the enzyme, and could impact the ability/speed of the enzyme to reactivate to its normal state (Wallace, 1992; Wallace and Kemp 1991). For instance, taxonomic differences in the electronic and steric properties of the esteratic site, nucleophilic strength of the enzyme center, the distance between the anionic and esteratic sites, and the electronic/steric properties of the anionic site may all impact the relative binding efficiency of the enzyme to the pesticide (O\u0026rsquo;Brien 1963; Monserrat and Bianchini 2001; Wallace, 1992; Wallace and Kemp 1991).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eBaseline levels of the enzyme can significantly vary depending on species, strains, age class, sex, season, reproductive and nutritional status, and disease state (Cowman and Mazanti, 2000; Hill 1988; Rattner and Fairbrother 1991).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eThere are specific differences in the genes that code for the cholinergic AChE enzyme. In vertebrate species, AChE is encoded by a single gene (Ace) resulting in a conserved enzyme across the taxonomic group (Lu et al., 2012; Taylor 2011). AChE is encoded in the Diptera suborder Brachycera (e.g., Drosophila, common house fly) by the gene Ace2, while in other insects both an Ace1 and Ace2 gene encode AChE (Lu et al., 2012). The Ace1 gene produces an AChE with a cysteine residue, which is not found in vertebrate AChE, or in the AChE from the Ace2 gene form. Acetylcholinesterase from Ace1 is associated with neurotransmissions within the insect, while AChE from the Ace2 gene is responsible for non-cholinergic activity such as embryonic development, growth, and reproduction (Lu et al., 2012).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eComparisons of susceptibility of Xenopus laevis and human forms of AChE to OP and carbamate pesticides found that the enzyme in frog embryos has a much higher resistance to these pesticides than the human form of the enzyme (Shapira et al., 1998).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eAcetylcholinesterase is found in presynaptic membranes of motor neurons in the spinal cord, cranial nerves within skeletal muscle, and preganglionic sympathetic and postganglionic parasympathetic neurons of vertebrates (Mileson et al., 1998). In invertebrates, AChE appears to be associated with sensory, brain, and other muscle activity (Fulton and Key, 2001; Habig and Di Giulio 1991; Mileson et al. 1998; Ware and Whitacre 2004).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\t\u003cli dir=\"ltr\"\u003e\r\n\t\t\u003cp dir=\"ltr\"\u003eIn plants, the function of AChE is not well understood, but levels of acetylcholine appear to be involved in the regulation of membrane permeability, and the ability of a leaf to unroll (Tretyn and Kendrick 1991).\u003c/p\u003e\r\n\t\t\u003c/li\u003e\r\n\t\u003c/ul\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003ch5 dir=\"ltr\"\u003eSex Applicability\u003c/h5\u003e\r\n\r\n\u003cp dir=\"ltr\"\u003eNo studies were located reporting significant differences in AChE activity between male and female organisms.\u003c/p\u003e\r\n","key_event_essentiality":"\u003cul\u003e\r\n\t\u003cli\u003eThere are numerous studies which have shown the blockage or reversibility of downstream events following the administration of pharmacological agents that either bypass acetylcholinesterase activity by directly activating acetylcholine receptors or that act as direct agonists or antagonists of the various types of acetylcholine receptors. Additionally, evidence from experiments with AChR morpholino antisense oligonucleotides have provided additional evidence for an essential role of acetylcholine receptor activation in mediating some of the downstream key events.\u003c/li\u003e\r\n\t\u003cli\u003eBased on the current information assembled for this AOP, the essentiality of the key events downstream of acetylcholine accumulation is less clear. While there are several key events that correspond with well known symptoms of acetylcholinesterase inhibition (as characterized through a number of nonadjacent KERs), it is presently unclear which of these are the major driver of lethality across different species. In humans, addressing respiratory failure is routinely used to prevent death from poisoning with chemicals that act on acetylcholinesterase. Data from ecological species suggest that other failure points could be equally important. Given the abundance of literature on acetylcholine signaling and adverse effects associated with acetylcholinesterase inhibition, this is an area of the AOP that warrants further development.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","weight_of_evidence_summary":"\u003cul\u003e\r\n\t\u003cli\u003eThere is strong evidence that the initial inhibition of the AChE enzyme is required prior to triggering key events that lead to the adverse outcome of mortality (See US EPA 2006; Grue and Shipley 1984).\u003c/li\u003e\r\n\t\u003cli\u003eThere is strong empirical evidence linking the key events, beginning with the molecular initiating event; AChE inhibition, followed by an increase in the acetylcholine at synapses of muscarinic and nicotinic receptors, and subsequent physiological and biochemical response resulting in cholinergic activity resulting in the death of the organism.\u003c/li\u003e\r\n\t\u003cli\u003eStrong evidence based on measured AChE inhibition and statistically-derived acute endpoints (e.g., LC/LD50) demonstrate a correlation of increase in enzyme inhibition and increased lethality. The open literature has many studies reporting these effects across invertebrate and vertebrate species with examples presented below.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThe overall weight of evidence supporting the indirect relationship between AChE inhibition and mortality is very strong and there are many physiological activities associated with acetylcholine neurotransmission that are plausibly linked with organism survival. However, there remain significant gaps in the current AOP description regarding which specific intermediate events are primarily responsible for the toxicity observed. It is likely that it is a combination of these physiological responses rather than any one alone, and that the key driver is also species dependent.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","quantitative_considerations":"","optional_considerations":"","references":"\u003cul\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eAdejumo, D.O. and G.N. Egbunike. 2004. Changes in acetylcholinesterase activities in the developing and aging pig brain and hypophyses. Int. J. Agric. Rural. Dev. 5: 46-53.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eAnguiano, O.L., C.M. Montagna, M. Chifflet de Llamas, L. Gauna, and A.M. Pechen de D\u0026#39;Angelo. 1994. Comparative toxicity of parathion in early embryos and larvae of the toad, Bufo arenarum Hensel. Bull. Environ. Contam. Toxicol. 52(5): 649-655.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eBerman, H.A., M.M. Decker, and J. Sangmee. 1987. Reciprocal regulation of acetylcholinesterase and butyrylcholinesterase in mammalian skeletal muscle. Dev. Biol. 120(1): 154-161.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eCowman, D.F. and L.E. Mazanti. 2000. Ecotoxicology of \u0026ldquo;new generation\u0026rdquo; pesticides to amphibians. In: D.W. Sparling, G. Linder, and C.A. Bishop (Eds.), Ecotoxicology of Amphibians and Reptiles, pp 233-268, SETAC Press, Pensacola, FL.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eEcobichon, D.J. 2001. Toxic effects of pesticides. In: C.D. Klaassen (Ed.), Casarett and Doull\u0026rsquo;s Toxicology: The Basic Science of Poisons; Sixth Edition. (pp. 763-810). McGraw-Hill, New York, NY.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eFukuto, TR. 1990. Mechanism of action of organophosphorus and carbamate insecticides. Environ Health Perspect. 87:245-254.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eFulton, M.H. and P.B. Key. 2001. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorous insecticide exposure and effects. Environ. Toxicol. Chem. 20(1): 37-45.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eGrue, C.E., G.V.N. Powell, and N.L. Gladson. 1981. Brain cholinesterase (ChE) activity in nestling starlings: Implications for monitoring exposure of nestling songbirds to ChE inhibitors. Bull. Environ. Contam. Toxicol. 26: 544-547.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eGrue, C.E., W.J. Fleming, D.G. Busby, and E.F. Hill. 1983. Assessing hazards of organophosphate pesticides to wildlife. In: Transactions of the 48th North American Wildlife and Natural Resources Conference, The Wildlife Management Institute, Washington, DC. pp. 200-220.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eGrue, C.E. and B.K. Shipley. 1984. Sensitivity of nestling and adult starling to dicrotophos, and organophosphate pesticide. Environ. Res. 35:454-465.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eGrue, C.E., P.L. Gibert, and M.E. Seeley. 1997. Neurophysiological and behavioral changes in non-target wildlife exposed to organophosphate and carbamate pesticides: Thermoregulation, food consumption, and reproduction. Amer. Zool. 37: 369-388.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eHabig, C. and R.T. DiGiulio. 1991. Biochemical characteristics of cholinesterases in aquatic organisms. In: P. Mineau (Ed.), Cholinesterase-inhibiting Insecticides: Their Impact on Wildlife and the Environment. pp. 19-33. Elsevier, Amsterdam, The Netherlands.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eHill, E.F. 1988. Brain cholinesterase activity of apparently normal wild birds. J.Wild. Dis. 24(1): 51-61.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eKaranth, S. and Pope, C. 2003. Age-Related Effects of Chlorpyrifos and Parathion on Acetylcholine Synthesis in Rat Striatum. Neurotoxicol.Teratol. 25[5], 599-606.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eLassiter TL, Barone S Jr, Moser VC, Padilla S (1999) Gestational exposure to chlorpyrifos: Dose response profiles for cholinesterase and carboxylesterase activity. Tox. Sci. 52:92-100.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eLu, Y., Y. Park, X. Gao, X. Zhang, J. Yoo, Y.-P. Pang, H. Jiang, and K.Y. Zhu. 2012. Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum. Sci. Rep. 2(Article No. 288):1-7.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eLudke, J.L., E.F. Hill, and M.P. Dieter. 1975. Cholinesterase (ChE) response and related mortality among birds fed ChE inhibitors. Arch. Environ. Contam. Toxicol. 3(1): 1-21.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eMileson, B.E., J.E. Chambers, W.L. Chen, W. Dettbarn, M. Ehrich, A.T. Eldefrawi, D.W. Gaylor, K. Hamernik, E. Hodgson, A.G. Karczmar, S. Padilla, C.N. Pope, R.J. Richardson, D.R. Saunders, L.P. Sheets, L.G. Sultatos, and K.B. Wallace. 1998. Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol. Sci. 41: 8-20.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eMonserrat, J.M. and A. Bianchini. 2001. Anticholinesterase effect of eserine (physostigmine) in fish and crustacean species. Braz. Arch. Biol. Technol. 44(1): 63-68.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eMoser VC, Phillips PM, McDaniel KL. (2015) Assessment of biochemical and behavioral effects of carbaryl and methomyl in Brown-Norway rats from preweaning to senescence. Toxicology 331, 1-13.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eMoser VC. (2011) Age-related differences in acetylcholinesterase inhibition produced by organophosphorus and N-methyl carbamate pesticides, in Pesticides in the Modern World B Pests Control and Pesticides Exposure and Toxicity Assessment (Stoytcheva M, ed). pp 495-506, Intech, Rijeka, Croatia\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eO\u0026rsquo;Brien, R.D. 1963. Mode of action of insecticides: Binding of organophosphate to cholinesterases. Agric. Food Chem. 11(2): 163-166.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eParkash, R. and K. Kaur. 1982. Ontogeny of acetylcholinesterases in hybridizing Drosophila species. Proc. Indian Nat. Sci. Acad. B48(5): 659-666.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eRattner, B.A. and A. Fairbrother. 1991. Biological variability and the influence of stress on cholinesterase activity. In: P. Mineau (Ed.), Cholinesterase-inhibiting Insecticides: Their Impact on Wildlife and the Environment. (pp. 89-107). Elsevier, Amsterdam, The Netherlands.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eSatoh, TETSUO. 2006. \u0026ldquo;CHAPTER 8 - Global Epidemiology of Organophosphate and Carbamate Poisonings.\u0026rdquo; In Toxicology of Organophosphate \u0026amp; Carbamate Compounds, edited by Ramesh C. Gupta, 89\u0026ndash;100. Burlington: Academic Press.\u003ca href=\"https://doi.org/10.1016/B978-012088523-7/50009-0\"\u003e https://doi.org/10.1016/B978-012088523-7/50009-0\u003c/a\u003e.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eSchűűrmann G. 1992. Ecotoxicology and structure-activity studies of organophosphorus compounds. Rational Approaches to Structure, Activity, and Ecotoxicology of Agrochemicals, CRC Press, Boca Raton, FL, USA pp 485-541\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eShapira, M., S. Seidman, N. Livni, and H. Soreq. 1998. In vivo and in vitro resistance to multiple anticholinesterases in Xenopus laevis tadpoles. Toxicol. Lett. 102-103: 205-209.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eSogob MA, Vilanova E. 2002. Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis. Toxicol Lett 128:215-228.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eStenersen, J. 2004. Specific enzyme inhibitors. In: Chemical Pesticides: Mode of action and toxicology. (41 p). CRC Press, Boca Raton, FL.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eTaylor, P. 2011. Anticholinesterase agents. In: L.J. Brunton (Ed.), Goodman and Gilman\u0026rsquo;s The Pharmacological Basis of Therapeutics; 12th Edition. (pp. 255-276). McGraw Hill, New York, NY. (Accessed from the web:\u003ca href=\"http://accessmedicine.com/resourceTOC.aspx?resourceID=651\"\u003e http://accessmedicine.com/resourceTOC.aspx?resourceID=651\u003c/a\u003e).\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eTretyn, A. and R.E. Kendrick. 1991. Acetylcholine in plants: Metabolism and mechanism of action. Bot. Rev. 57(1): 33-73.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eTsim, K.W.K., W.R. Randall, and E.A. Barnard. 1988. Synaptic acetylcholinesterase of chicken muscle changes during development from a hybrid to a homogenous enzyme. EMBO J 7(8): 2451-2456.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eU.S. Environmental Protection Agency (U.S. EPA). 2006. Organophosphorus Cumulative Risk Assessment \u0026ndash; 2006. Update. U.S. EPA, Office of Pesticide Programs, Washington, DC. 522 p.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eWallace, K.B., and J.R. Kemp. 1991. Species specificity in the chemical mechanisms of organophosphorus anticholinesterase activity. Chem. Res. Toxicol. 4: 41-49.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eWallace, K.B. 1992. Species-selective toxicity of organophosphorus insecticides: A pharmacodynamics phenomenon. In: J. E. Chambers and P. E. Levi, (Eds.), Organophosphates\u0026mdash; Chemistry, Fate, and Effects. pp. 79-105. Academic Press, San Diego.\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\t\u003cli dir=\"ltr\"\u003e\r\n\t\u003cp dir=\"ltr\"\u003eWare, G.W. and D.M. Whitacre. 2004. An Introduction to Insecticides. In: E. B. Radcliffe,W. D. Hutchison and R. E. Cancelado [Eds.], Radcliffe\u0026#39;s IPM World Textbook. 34 pp. University of Minnesota, St. Paul, MN. (Accessed from the web: URL:\u003ca href=\"http://ipmworld.umn.edu\"\u003e http://ipmworld.umn.edu\u003c/a\u003e).\u003c/p\u003e\r\n\t\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","overall_assessment":"","background":"","user_defined_mie":"12: Acetylcholinesterase (AchE) Inhibition","user_defined_ao":"351: Increased Mortality and 360: Decrease, Population trajectory","oecd_project":"1.3","oecd_status_id":4,"graphical_representation_image_uid":"2019/12/20/9fswvuyk6n_AChE_AcuteMortality.png","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2019-12-23T11:16:32.000-05:00","development_strategy":"","known_modulating_factors":"\u003cdiv\u003e\r\n\u003ctable class=\"table table-bordered table-fullwidth\"\u003e\r\n\t\u003cthead\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003cth\u003eModulating Factor (MF)\u003c/th\u003e\r\n\t\t\t\u003cth\u003eInfluence or Outcome\u003c/th\u003e\r\n\t\t\t\u003cth\u003eKER(s) involved\u003c/th\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/thead\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\u003c/div\u003e\r\n","assigned_license_id":14,"handbook_id":1,"project_129":false},{"id":17,"title":"Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress during brain development leads to impairment of learning and memory","short_name":"Oxidative stress and Developmental impairment in learning and memory","corresponding_author_id":245,"abstract":"\u003cp style=\"text-align:justify\"\u003eThis Adverse Outcome Pathway (AOP) describes the linkage between binding to sulfhydryl(SH)-/seleno-proteins involved in protection against oxidative stress and impairment in learning and memory, the Adverse Outcome (AO). Binding to SH-/ seleno-proteins involved in protection against oxidative stress has been defined as the Molecular Initiating Event (MIE). Production, binding and degradation of Reactive Oxygen Radicals (ROS) are tightly regulated, and an imbalance between production and protection may cause oxidative stress, which is common to many toxicity pathways. Oxidative stress may lead to an imbalance in glutamate neurotransmission, which is involved in learning and memory. Oxidative stress may also cause cellular injury and death. During brain development and in particular during the establishment of neuronal connections and networks, such perturbations may lead to functional impairment in learning and memory. Neuroinflammation (Resident cell activation; Increased pro-inflammatory mediators) is triggered early in cell injury cascades and is considered as an exacerbating factor. The weight-of-evidence supporting the relationship between the described key events is based mainly on developmental effects observed after an exposure to the heavy metal, mercury, known for its strong affinity to many SH-/seleno-containing proteins, but in particular to those having anti-oxidant properties, such as glutathione (GSH). The overall assessment of this AOP is considered as strong, based on the biological plausibility, the empirical support and on the essentiality of the Key Events (KEs), which are moderate to strong, since blocking, preventing or attenuating an upstream KE is mitigating the downstream KE. The gap of knowledge is mainly due to limited quantitative evaluations, impeding thus the development of predictive models.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2025-07-02T02:28:06.000-04:00","status_id":1,"authors":"\u003cp\u003eFlorianne Tschudi-Monnet, Department of Biological Sciences, University of Lausanne, Switzerland, and Swiss Centre for Applied Human Toxicology (SCAHT), Florianne.Tschudi-Monnet@unil.ch\u003c/p\u003e\r\n\r\n\u003cp\u003eMarie-Gabrielle Zurich, Department of Biological Sciences, University of Lausanne and SCAHT, Switzerland, mzurich@unil.ch\u003c/p\u003e\r\n\r\n\u003cp\u003eCarolina Nunes, Department of Biological Sciences, University of Lausanne, Switzerland, carolina.nunes@unil.ch\u003c/p\u003e\r\n\r\n\u003cp\u003eJenny Sandstr\u0026ouml;m, SCAHT, Switzerland, jsm.sandstrom@gmail.com\u003c/p\u003e\r\n\r\n\u003cp\u003eRex FitzGerald, SCAHT, Switzerland, rex.fitzgerald@unibas.ch\u003c/p\u003e\r\n\r\n\u003cp\u003eMichael Aschner, Albert Einstein College of Medecine, New York, USA, michael.aschner@einstein.yu.edu\u003c/p\u003e\r\n\r\n\u003cp\u003eJoao Rocha, Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, Brazil, jbtrocha@gmail.com\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eThe authors of KEs AOPwiki ID 1392 (oxidative stress), 55 (Cell injury/death), 386 (Decrease network function), of the AO (Learning and memory, impairment), and of KER 359 (decrease network function leads to impairment in learning and memory) are greatly acknowledged.\u003c/p\u003e\r\n\r\n\u003cp style=\"margin-left:36.0pt\"\u003e\u0026nbsp;\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-family:Arial,Helvetica,sans-serif\"\u003e\u003cspan style=\"font-size:12pt\"\u003eThis AOP is mainly focused on the developmental period, although it cannot be excluded that long-term exposure in adult may trigger a similar cascade of KEs leading also to impairment in learning and memory, as observed in neurodegenerative diseases such as Alzheimer\u0026#39;s disease (Mutter et al., 2004). While no specific sex differences have been analyzed/described for most KEs, Curtis and coworkers (2010) observed a higher level of TNF-\u003c/span\u003e\u003cspan style=\"font-size:12pt\"\u003ea\u003c/span\u003e\u003cspan style=\"font-size:12pt\"\u003e in hippocampus of male prairie wolf than in female, both treated for 10 weeks with inorganic mercury, in the form of HgCl\u003csub\u003e2\u003c/sub\u003e; whereas Zhang and coworkers (2013) found a higher neuroinflammatory response associated with altered social behavior in female mice offspring than in male, following gestational exposure to HgCl\u003csub\u003e2\u003c/sub\u003e. However, after developmental methylmercury exposure, long-lasting behavioral alterations were more prominent in males (Ceccatelli et al., 2013; Castoldi et al., 2008b). These discrepancies may be due to sex differences in kinetics or susceptibility (Vahter et al., 2006).\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n","key_event_essentiality":"\u003ctable align=\"left\" cellspacing=\"0\" class=\"MsoTable15Grid5DarkAccent1\" style=\"border-collapse:collapse; border:none\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd rowspan=\"2\" style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:none; border-top:1px solid white; height:30px; vertical-align:top; width:145px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:30px; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:4px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDefining Question\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:30px; vertical-align:top; width:125px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eHigh (Strong)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:30px; vertical-align:top; width:111px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eModerate\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:30px; vertical-align:top; width:114px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eLow (Weak)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:4px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eAre downstream KEs and/or the AO prevented if an upstream KE is blocked?\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:125px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eDirect evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g. stop/reversibility studies, antagonism, KO models, etc.)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:111px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eIndirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:114px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eNo or contradictory experimental evidence on the essentiality of any of the KEs\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDecreased protection against oxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:4px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: The fact that a decrease in anti-oxidant properties causes oxidative stress is well accepted. In addition, experimental evidences of knocking out proteins involved in protection against oxidative stress incresed the susceptibilty to oxidative stress.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eOxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:4px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: The deleterious consequences of oxidative stress are well accepted in various animal models. Oxygen radical scavengers, such as glutathione, catalase, selenium and cysteine can block the deleterious effects of oxidative stress.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE3 \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eGlutamate dyshomeostasis\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:4px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: Glutamate is the main excitatory transmitter, and is involved in memory processes, it is well accepted that perturbation of glutamate homeostasis has deleterious functional consequences. Disruption of glutamate signaling is thought to play a role, at least in part, in the etiology underlying several neurodevelopmental disorders, including memory dysfunction.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eCell Injury/death, increased\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:4px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: Cell injury/death is a highly converging node in AOPs. Decrease in synaptic connectivity or cell loss will in turn induce perturbations in the establishment of neuronal connections and trigger inflammatory responses, which through a feedback loop can exacerbate this KE. Therefore, prevention of cell injury/death by anti-oxidant or by inhibitors of NMDA receptors prevents the downstream KEs.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eNeuroinflammation\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE5\u0026#39; Tissue resident cell activation\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE5\u0026#39;\u0026#39; Pro-inflammatory mediators, increased\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:4px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMODERATE\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: It is widely accepted in different experimental animal models that the use of minocycline, an antibiotic, which blocks microglial reactivity has protective effects, as have other interferences with any inflammatory mediators. However, we rate the essentiality of this KE as moderate given the complexity of the neuroinflammatory response, having either protective/reparative or aggravating consequences,\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:145px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDecreased network formation and function\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:4px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:351px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: Glutamate neurotransmission is an important mechanism underlying memory function (for review: Featherstone, 2010). During brain development, glutamate has also trophic effects, by stimulating BDNF production or through the activation of the different glutamate receptors. The\u0026nbsp;trophic\u0026nbsp;effect of glutamate receptor activation is developmental stage-dependent and may play an important role in determining the selective survival of neurons that made proper connections (Balazs, 2006).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eAO \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eImpairment of learning and memory\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:4px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: Impairment in learning and memory is a converging KE in several AOPs related to brain development. Regarding this AOP and its chemical initiators, it was shown that the neurocognitive domain, in particular dentate gyrus, hippocampus and cortex are susceptible to the neurotoxicity of mercury in the developing brain (Sokolowski et al., 2011, 2013; Ceccatelli et al., 2013). Chronic, low-dose prenatal MeHg exposure from maternal consumption of fish has been associated with endpoints of neurotoxicity in children, including poor performance on neurobehavioral tests, particularly on tests of attention, fine-motor function, language, visual-spatial abilities (e.g., drawing), and verbal memory (NRC, 2000). Prenatal MeHg exposure is associated with childhood memory and learning deficits, particularly visual memory performance in school-aged children (Orenstein, 2014).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003e\u003cstrong\u003eDose-response and temporal concordance of KEs\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThere is no study where all KEs are measured simultaneously after exposure to several doses, impeding a dose-response and concordance analysis. In one single study (in blue in the table), three downstream KEs were measured following pre-natal exposure to methylmercury. Comparisons of all animal studies show that doses used are ranging from 0.5 - 5 mg/kg; but dose-response was seldom performed. In these studies, the time (pre-natal, post-natal, lactation,...) and duration of exposure are quite diverse and no analysis of brain mercury content was made, so it is not possible to compare doses between studies. Therefore, based on the present data, it is impossible to define whether KEs up occur at lower doses and earlier time points than KEs down.\u003c/p\u003e\r\n\r\n\u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e studies, KEs up are often measured after acute exposure to high concentrations.\u003c/p\u003e\r\n\r\n\u003cp\u003eThe following table summarizes concentrations/doses, time, and duration of exposure for the various test systems and KEs.\u003c/p\u003e\r\n\r\n\u003ctable align=\"left\" cellspacing=\"0\" class=\"MsoTable15Grid5DarkAccent1\" style=\"border-collapse:collapse; border:none; width:605px\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:none; border-top:1px solid white; height:64px; width:141px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKEs\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:64px; width:220px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-203px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eIn vivo\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:64px; width:244px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eIn vitro\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:141px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eMIE\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eBinding to SH-/seleno-proteins\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:220px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:244px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eBinding of Hg to thiol groups and to various selenium-containing proteins:\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eGlutathione, thioredoxin reductase, thioredoxin, glutaredoxin, glutathione reductase was measured using purified proteins\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e(Carvahlo et al., 2008, 2011; Wiederhold et al., 2010; Sugiura et al., 1978; Arnold et al., 1986; Han et al., 2001; Qiao et al., 2017)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:141px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDecreased protection against oxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:220px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eCytoplasmic and nuclear TrxR and Cytoplasmic Gpx were reduced in cerebral and cerebellar cortex of 22 days-old offspring (Ruszkiewicz, 2016)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMale C57BL/6NJcl mice exposed to methylmercury (1.5 mg/kg/day for 6-weeks) (Fujimura, 2017)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eAdult male Sprague-Dawley rats exposed to methylmercury (1 mg/kg orally for 6 months) (Joshi, 2014)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eZebra fish brain exposed to Hg2+, MeHg 1.8 molar (measured in brain tissue), for 28 days (Branco, 2012)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003ePrenatal and postnatal exposure of mice to 40 ppm of HgCl\u003csub\u003e2\u003c/sub\u003e decreased the activity of catalase, thioredoxin reductase, Gpx, superoxide dismutase (Malqui et al., 2017)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:244px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMouse primary cortical cultures exposed to 5 mM of methylmercury for 24h (Rush, 2012)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMeHg inhibits ex vivo rat thioredoxin reductase; IC50 0.158 \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026mu;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eM (cerebral) (Wagner et al., 2010)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHuman neuroblastoma cells (SH-SY5Y)exposed to 1 \u0026micro;M of methylmercury for 6 or 24 h (Branco, 2017; Franco, 2009)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:141px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eOxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:220px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMale C57BL/6NJcl mice exposed to methylmercury (1.5 mg/kg/day for 6-weeks) (Fujimura, 2017)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eAdult male Sprague-Dawley rats exposed to methylmercury (1 mg/kg orally for 6 months) (Joshi, 2014)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eAdult male Sprague-Dawley rats exposed to methylmercury (1 mg/kg orally for 6 months) (Joshi, 2014)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eZebra fish brain exposed to Hg2+, MeHg 1.8 molar (measured in brain tissue), for 28 days (Branco, 2012)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003ePrenatal and postnatal exposure of mice to 40 ppm of HgCl\u003csub\u003e2\u003c/sub\u003e caused oxidative stress evaluated by increased lipid peroxidation (Malqui et al., 2017)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:244px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMouse primary cortical cultures exposed to 5 mM of methylmercury for 24h (Rush, 2012)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMethylmercury (2-10 \u0026micro;M) in synaptic vesicles isolated from rat brain (with LD\u003csub\u003e50\u003c/sub\u003e at 50 \u0026micro;M) (Porciuncula et al., 2003)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHuman neuroblastoma cells (SH-SY5Y)exposed to 1 \u0026micro;M of methylmercury for 6-24 h (Franco, 2009)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:141px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE3 \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eGlutamate dyshomeostasis\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:220px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRat Young (3-4 weeks) dosed with acrylamide by gavage (5, 15, 30 mg/kg, 5 applications per week during 4 weeks) (Tian, 2018)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMicrodialysis probe in adult Wistar rats showed that acute exposure to methylmercury (10, 100 mM) induced an increase release of extracellular glutamate (9.8 fold at 10 mM and 2.4 fold at 100 mM). This extracellular glutamate level remained elevated at least 90 min (Juarez et al., 2002)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:244px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMouse astrocytes, neurons in mono- or co-cultures exposed to methylmercury 1-50 \u0026micro;M for 24h (Morken, 2005)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMethylmercury (2-10 \u0026micro;M) in synaptic vesicles isolated from rat brain (with LD\u003csub\u003e50\u003c/sub\u003e at 50 \u0026micro;M) (Porciuncula et al., 2003\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:141px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eCell Injury/death, increased\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:220px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRat, perinatal exposure to methylmercury (GD7-PD21, i.e. 35 days) 0.5 mg/kg bw/day in drinking water (Roda et al., 2008)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRat Young (3-4 weeks) exposed to acrylamide by gavage (5, 15, 30 mg/kg, 5 applications per week during 4 weeks) (Tian, 2018)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003ePregnant rat exposed to methylmercury (1.5 mg/kg orally) from GD5 till parturition (Jacob, 2017)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:244px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMouse astrocytes, neurons in mono- or co-cultures exposed to methylmercury 1-50 \u0026micro;M for 24h (Morken, 2005)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:141px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eNeuroinflammation\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE5\u0026#39; Tissue resident cell activation\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE5\u0026#39;\u0026#39; Pro-inflammatory mediators, increased\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:220px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRat, perinatal exposure to methylmercury (GD7-PD21, i.e. 35 days) 0.5 mg/kg bw/day in drinking water (Roda et al., 2008)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMonkeys, 6,12,18 months oral exposure 50 mg/kg bw (Charleston et al., 1996)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:244px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e3D rat brain cell cultures 10 day treatmentHgCl\u003csub\u003e2\u003c/sub\u003e 10\u003csup\u003e-9\u003c/sup\u003e-10\u003csup\u003e-6\u003c/sup\u003eM\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMeHgCl 10\u003csup\u003e-9\u003c/sup\u003e-3x10\u003csup\u003e-7\u003c/sup\u003eM (Monnet-Tschudi et al., 1996; Eskes et al., 2002)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:141px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDecreased network formation and function\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:220px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMice dosed during postnatal week 1-3 with subcutaneous 2-5 mg mercury chloride/kg/once per week (Eddins et al., 2008)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003ePregnant rat dosed on GD 15 with 8 mg/kg of methylmercury by gavage. Offsprings were tested at day 16, 21 and 60. (Cagiano et al., 1990)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003ePregnant rat exposed to methylmercury (1.5 mg/kg orally) from GD5 till parturition (Jacob, 2017)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:244px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:141px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eAO \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eImpairment of learning and memory\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:220px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMice dosed during postnatal week 1-3 with subcutaneous 2-5 mg mercury chloride/kg/once per week (Eddins et al., 2008)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003ePregnant rat dosed on GD 15 with 8 mg/kg of methylmercury by gavage. Offsprings were tested at day 16, 21 and 60 (Cagiano et al., 1990)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003ePregnant rat exposed to methylmercury (1.5 mg/kg orally) from GD5 till parturition (Jacob, 2017)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003ePregnant mice received 0.5 mg methylmercury/kg/day in drinking water from gestational day 7 until day 7 after delivery. Offspring behavior was monitored at 5-15 and 26-36 weeks of age (Onishchenko et al., 2007)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eBalb mice exposed to methylmercury in diet (low dose: 1.5 mg/kg; high dose: 4.5 mg/kg) during 11 weeks (6 weeks prior mating, 3 weeks during gestation and 2 weeks post-partum). Offsprings tested at PD 15 showed an accumulation of Hg in brain (0.08 mg/kg for low dose and 0.25 mg/kg for the high dose) (Glover et al., 2009)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003ePrenatal and postnatal exposure of mice to 40 ppm of HgCl\u003csub\u003e2\u003c/sub\u003e caused impairment of memory (object recognition, Y maze) Malqui et al., 2017)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:244px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMaternal peripartum hair mercury level was measured to assess prenatal mercury exposure. The concentrations of mercury was found in the range of 0.3-5.1 \u0026micro;g/g, similar to fish-eating population in US. Statistical analyses revealed that each ug/g increase in hair Hg was associated with a decrement in visual memory, learning and verbal memory \u003cstrong\u003e(\u003c/strong\u003eOrenstein et al., 2014)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eEpidemiological studies in the Faroe Islands revealed that mercury exposure through fish consumption (maternal hair conc. 10 ug/g) dysfunctions in memory, language and attention at age 7 (Grandjean et al., 1997; Debes et al., 2006)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cspan style=\"font-family:Arial,Helvetica,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:12pt\"\u003eBiological Plausibility and Empirical Support of the KERs\u003c/span\u003e\u003c/strong\u003e\u0026nbsp; \u003c/span\u003e\u003c/p\u003e\r\n\r\n\u003ctable cellspacing=\"0\" class=\"MsoTable15Grid5DarkAccent1\" style=\"border-collapse:collapse; border:none; width:841px\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:none; border-top:1px solid white; height:170px; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKERs\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:170px; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDefining Question\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eIs there a mechanistic (i.e. structural or functional) relationship between KEup and KEdown consistent with established biological knowledge?\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:170px; vertical-align:top; width:130px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:13px; margin-right:-47px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eHigh (Strong)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:4px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eExtensive understanding of the KER based on extensive previous documentation and broad acceptance\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:170px; vertical-align:top; width:138px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:7px; margin-right:-47px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eModerate\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:7px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eThe KER is plausible based on analogy to accept biological relationship but scientific understanding is not completely established\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:170px; vertical-align:top; width:280px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px; margin-right:-47px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eLow (Weak)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eThere is empirical support for a statistical association between KEs but the structural or functional relationship between them is not understood\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eMIE to KE Decrease protection against oxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMODERATE\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: Thiol- and selenol containing proteins, which mainly belong to the anti-oxidant protections, have a high affinity for binding soft metals such as mercury (Farina, 2011). Binding to these thiol/sulfhydryl/SH/SeH groups results in structural modifications affecting the catalytic capacity, and thereby reducing the capacity to neutralize ROS. However, binding to other SH/SeH groups of other proteins not involved in protection against oxidative stress can occur and trigger other neurotoxicity pathways. Alternatively, binding to SH groups of electrophilic compounds may also induce cyto-protective reactions (e.g. via Nrf2).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE Decrease protection against oxidative stress to KE Oxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. Several studies have shown depletion of GSH, the main anti-oxidant, and an increase in oxidative stress following methylmercury or mercury chloride exposures (Meinerz, 2011; Rush, 2012; Agrawal, 2015). Protection against oxidative stress was observed by supplementation with diphenyl selenide (Meinerz, 2011) or by glutathione ester (Rush, 2012). \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eLimited conflicting data.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE Oxidative stress to KE Glutamate (Glu) dyshomeostasis\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eLOW\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE:\u0026nbsp;Glutamate transport is driven by the Na\u003csup\u003e+\u003c/sup\u003e ion gradient, which is dependent on the Na/K ATPase, which, in turn, requires energy. Glutamate enters the cells accompanied by 2 Na\u003csup\u003e+ \u003c/sup\u003eand one H\u003csup\u003e+\u003c/sup\u003e.\u0026nbsp; Perturbations of energy metabolism such as mitochondrial dysfunction and increased production of ROS will lead to glutamate dyshomeostasis, due to the indirect coupling of glutamate transporters with ATP level, and to the important role of glutamate transporters in glutamate homeostasis. (Boron and Boulpaep, 2003). Methylmercury was shown to inhibit both the H\u003csup\u003e+\u003c/sup\u003e-ATPase activity and vesicular glutamate uptake (Porciuncula et al., 2003). As, on one hand, ROS production can interfere with glutamate uptake, and on the other hand, glutamate accumulation leads to excitotoxicity and ROS production, the exact sequence of the KER is difficult to assess. But the fact that both KEs are involved in mercury-induced neurotoxicity is broadly accepted (Farina et al., 2011; Antunes dos Santos et al., 2016; Morris et al., 2017; Kern et al., 2016).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE Glutamate dyshomeostasis to KE Cell injury/death\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: Glutamate dyshomeostasis, in particular excess of glutamate in the synaptic cleft, leads to overactivation of ionotropic glutamate receptors, referred to as excitotoxicity. This, in turn, will cause cell injury/death via ROS production. This KER is also inherent to the developing brain, where glutamate ionotropic receptors are expressed early in various neural cells and when NMDA receptors are expressed in neurons. There is empirical support for all three chemical initiators (mercury, acrylamide, acrolein). In addition, several experiments aiming at blocking glutamate excitotoxicity and the resulting ROS production are protective for cell injury/death. \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eLimited conflicting data.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE Cell injury/death to KE Neuroinflammation\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMODERATE\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: It is widely accepted that cell/neuronal injury and death lead to neuroinflammation (microglial and astrocyte reactivities) in adult brain, and in the developing brain, where neuroinflammation was observed after cell injury/death induced by excitotoxic lesions (Acarin et al., 1997; Dommergues et al., 2003). Empirical support is available for all three chemical initiators (mercury, acrylamide, acrolein). \u0026nbsp;Few experiments, showing a protection when blocking any feature of neuroinflammation have been described. There are some contradicting data showing an absence of neuroinflammatory response despite the occurrence of mercury-induced apotosis and slight behavioral alterations.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE Neuroinflammation to KE Cell injury/death\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eMODERATE\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: In vitro co-culture experiments have demonstrated that reactive glial cells (microglia and astrocytes) can kill neurons via the release of pro-inflammatory cytokines, such as TNF-a, IL-1b and IL-6 and/or ROS/RNS (Chao et al., 1995; Brown and Bal-Price, 2003; Kraft and Harry, 2011; Taetzsch and Block, 2013) and that interventions aiming at blocking these inflammatory biomolecules can rescue the neurons (Yadav et al., 2012; Brzozowski et al., 2015). Several reports showed that modulating mercury or acrylamide-induced neuroinflammation was protective for neurons. Because of the complexity of the neuroinflammatory response, that can have neuroprotective or neurodegenerative consequences depending on the duration, local environment or still unknown factors, the rating of this KER was kept as moderate. The vicious cycle between cell injury/death and neuroinflammation is well known and was described in other AOPs. Neuroinflammation could be considered as a modulating factor, but because of the numerous inhibiting experiments, it is considered as an essential KE. Some conflicting data due to the dual role of some inflammatory mediators have been reported.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE Cell injury/death to KE Decreased network formation and function\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:548px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: Neuronal network formation and functional crosstalk are established via synaptogenesis. It was shown that under physiological conditions components of the apoptotic machinery in the developing brain regulate synapse formation and neuronal connectivity (Dekkers et al., 2013). The brain\u0026rsquo;s electrical activity dependence on synapse formation is critical for proper neuronal communication. Glial cells are also involved in the establishment and stabilization of the neuronal network. Extensive experimental support for the adverse effects of mercury on synaptogenesis exist, establishing a strong link between mercury-induced apoptosis and/or neuronal loss and perturbations in a number of neurotransmitter systems (Jacob, 2017; Bridges, 2017) and perturbations of functionality (Falluel-Morel, 2007; Ferraro, 2009; Teixera, 2014; Onishchenko, 2007). \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eLimited protective experiments and conflicting data reported.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE Decreased network formation and function to AO Impairment in learning and memory\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: A review on the Morris water maze (MWM) (Morris, 1981), as an investigative tool of spatial learning and memory in laboratory rats (Vorhees and Williams, 2006) pointed out that perturbed neuronal networks rather than neuronal death per se in certain regions is responsible for the impairment in MWM performance. Functional integrated neural networks that involve the coordination action of different brain regions are consequently important for spatial learning and memory performance (D\u0026#39;Hooge and De Deyn, 2001). Broad empirical support showing mercury-induced effects on learning and memory as consequence of network disruption (Sokolowski et al. 2013; Eddins et al., 2008; Glover et al., 2009). Similar observations were made in humans (Orenstein et al., 2014; Yorifuji et al., 2011).\u0026nbsp; Interestingly, behavioral alterations were detected long time after exposure (delayed effects). Few conflicting data have been reported, but other behavioral deficits, such as alterations in motor activity and increased anxiety suggest that systems other than hippocampus-related learning and memory are also affected.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:151px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:8px; margin-right:-1px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE oxidative stress to KE Cell injury/death\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:9px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eHIGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:6px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eRATIONALE: The central nervous system is especially vulnerable to free radical damage since it has a high oxygen consumption rate, an abundant lipid content and reduced levels of antioxidant enzymes (Coyle and Puttfarcken, 1993; Markesbery, 1997). The developing nervous system is particularly vulnerable to chemical insults (Grandjean and Landrigan, 2014). One reason for this higher vulnerability is the incapacity of immature neural cells to cope with oxidative stress by increasing glutathione (GSH) production (Sandstr\u0026ouml;m et al., 2017a). Broad empirical support for mercury and acrylamide showing an association between increased ROS production and/or decreased protection against oxidative stress and apoptosis and/or necrosis (Lu \u003cem\u003eet al.\u003c/em\u003e, 2011; Sarafian \u003cem\u003eet al.\u003c/em\u003e, 1994; Allam \u003cem\u003eet al.\u003c/em\u003e, 2011; Lakshmi \u003cem\u003eet al.\u003c/em\u003e, 2012). Anti-oxidant treatments proved to be protective. Few conflicting data, except a mercury-induced upregulation of GSH level and GR activity as an adaptive mechanism following lactational exposure to methylmercury (10 mg/L in drinking water) associated with motor deficit, suggesting neuronal impairment (Franco et al., 2006).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","quantitative_considerations":"\u003cp\u003eSome quantitative relationships have been described between the upstream early KEs (MIE, oxidative stress, Cell injury/death), although the diversity of test systems and posology (dosing/exposure amount and duration) hampers comparison between studies. It is more difficult to evaluate quantitative relationships between later downstream KEs, such as Neuroinflammation and Decreased Network Function. Neuroinflammation is a complex adaptive mechanism which is not yet completely understood; it can have neuroprotective or neurodegenerative consequences, depending on triggering signals, duration, microenvironment or other unknown influences, which may determine the outcome of the neuroinflammatory process. Decreased network function is currently difficult to quantify because quantitative technologies for mapping and understanding of brain networks (and their plasticity) are still under development.\u003c/p\u003e\r\n\r\n\u003cp\u003eOptimally, we would like data from a single type of test system showing that exposure to stressor, e.g. mercury, is correlated with changes in all KEs. Such models are emerging, using cells of human origin (Pamies et al., 2016; Sandstr\u0026ouml;m et al., 2017b; Fritsche et al., 2017) and/or non-mammalian models, such as zebrafish (Geier et al., 2018; Padilla et al., 2018) and will allow in the future generation of quantitative data which may be used for \u003cem\u003ein silico\u003c/em\u003e hazard prediction.\u003c/p\u003e\r\n\r\n\u003cp\u003eSummary table of Quantitative Evaluations\u003c/p\u003e\r\n\r\n\u003ctable align=\"left\" cellspacing=\"0\" class=\"MsoTable15Grid5DarkAccent1\" style=\"border-collapse:collapse; border:none\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:none; border-top:1px solid white; height:47px; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKEs\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:47px; width:174px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-203px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eMethylmercury\u003cstrong\u003e (\u003c/strong\u003eMeHg,\u003cstrong\u003e \u003c/strong\u003eCH\u003csub\u003e3\u003c/sub\u003eHg)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:47px; width:182px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:47px; vertical-align:top; width:211px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-6px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:47px; vertical-align:top; width:229px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-6px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e5 \u0026micro;M\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003emouse brain \u003cem\u003ein vitro\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e(Rush, 2012)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e15\u0026ndash;30 \u0026micro;M\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003emouse brain, after 40 mg/L in drinking water for 21 days\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e(Glaser, 2013)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e1 \u0026micro;M\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003emouse cerebral cortex ex vivo after oral dosing\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e(Lu et al 2011; conc. from Huang et al 2008)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e17-24, 75-\u0026micro;M (rat cerebral cortex \u003cem\u003eex vivo\u003c/em\u003e after 4w ip dosing)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e4w\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e(Xu, 2012; Liu 2013; Feng, 2014)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDecreased protection against oxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eGSH reduced 80% of control\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e24h\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eCortical mitochondrial GPx activity decreased (70% of control), GR increased (170% of control)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eGSH decreased (ca 50% of control)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e7 weeks\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eAntioxidants NPSH, SOD, GSH-Px decreased (ca 80% and 50% of control)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eOxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eROS increased 120-150% of control\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e24h\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eCortical mitochondrial TBA-RS increased (ca 140% of control) and complex I, II-III, and IV activity decreased (ca 50% of control).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eBrain 8-OHdG content increased (ca 400% of control).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eLPO increased (ca 200% of control)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e7 weeks\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eROS (DCF) increased (190 and 400% of control at 22,87 \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026mu;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eM)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE3 \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eGlutamate dyshomeostasis\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eGlutamine synthetase decreased (80 and 50% of control at 24,89 \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026mu;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eM)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eGlutamate content increased (100 and 120% of control at 24,89 \u0026micro;M)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eGlutamine content decreased (80 and 50% of control at 24,89 \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e\u0026mu;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eM)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eCell Injury/death, increased\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eApoptosis-related gene expression: Bcl-2 decreased, ca 50% of control; Bax, Bak, p53, caspase-3,-5,-7 increased, ca 200-350% of control\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e7 weeks\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eApoptosis increased dose-dependently (300 and 853% of control at 24,89 \u0026micro;M).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e8-OHdG expression increased (200 and 450% of control at 24,89 \u0026micro;M)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eNeuroinflammation\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDecreased network formation and function\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eAO \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eImpairment of learning and memory\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003ctable align=\"left\" cellspacing=\"0\" class=\"MsoTable15Grid5DarkAccent1\" style=\"border-collapse:collapse; border:none\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:none; border-top:1px solid white; height:47px; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKEs\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:47px; width:174px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-203px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eMercuric chloride\u003cstrong\u003e (\u003c/strong\u003eHgCl\u003csub\u003e2\u003c/sub\u003e)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:47px; width:182px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:47px; vertical-align:top; width:211px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-6px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:47px; vertical-align:top; width:229px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-6px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e6 \u0026micro;M\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003erat brain, 1.13 \u0026micro;g Hg/g\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e6 mo\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e(Agrawal, 2015)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e0.1-100 \u0026micro;M cultured mouse cerebellar granule cells\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e10 min\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003e(Fonfria, 2005)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDecreased protection against oxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eBlood GSH decreased (ca 90% of control)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eOxidative stress\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE3 \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eGlutamate dyshomeostasis\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eGlutamate (3H-aspartate) uptake inhibited (IC50 3.5 uM).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eGlutamate release stimulated (47% of total endogenous glutamate at 10 \u0026micro;M)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eCell Injury/death, increased\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eSerum AST increased (ca 140% of control).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eCell viability (MTT) decreased (ca 10% of control at 10 \u0026micro;M)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eNeuroinflammation\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eKE6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eDecreased network formation and function\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:black\"\u003eBrain noradrenaline and dopamine content decreased (ca 30% of control).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd style=\"background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px\"\u003e\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eAO \u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp style=\"margin-left:2px\"\u003e\u003cspan style=\"font-size:12pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Calibri\u0026quot;,sans-serif\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size:9.0pt\"\u003e\u003cspan style=\"font-family:\u0026quot;Arial\u0026quot;,sans-serif\"\u003e\u003cspan style=\"color:white\"\u003eImpairment of learning and memory\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp style=\"margin-right:-7px\"\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"2\" style=\"background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd style=\"background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px\"\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n","optional_considerations":"\u003cul\u003e\r\n\t\u003cli\u003eContribution to the network of KEs/AOPs on Developmental Neurotoxicity (DNT)\u003c/li\u003e\r\n\t\u003cli\u003eGenerating quantitative data by measuring all KEs in a single model after repeated/long term exposure to a wide concentration range of the chemical initiators to facilitate the development of computational predictive approaches\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","references":"\u003cp\u003eAcarin, L., B. Gonz\u0026aacute;lez, B. Castellano and A. J. Castro (1997). \u0026quot;Quantitative analysis of microglial reaction to a cortical excitotoxic lesion in the early postnatal brain.\u0026quot; \u003cu\u003eExp.Neurol.\u003c/u\u003e \u003cstrong\u003e147\u003c/strong\u003e: 410-417.\u003c/p\u003e\r\n\r\n\u003cp\u003eAgrawal, S., P. Bhatnagar and S. J. Flora (2015). \u0026quot;Changes in tissue oxidative stress, brain biogenic amines and acetylcholinesterase following co-exposure to lead, arsenic and mercury in rats.\u0026quot; Food Chem Toxicol 86: 208-216.\u003c/p\u003e\r\n\r\n\u003cp\u003eAllam,\u0026nbsp; a \u003cem\u003eet al.\u003c/em\u003e (2011) \u0026lsquo;Prenatal and perinatal acrylamide disrupts the development of cerebellum in rat: Biochemical and morphological studies.\u0026rsquo;, \u003cem\u003eToxicology and industrial health\u003c/em\u003e, 27, pp. 291\u0026ndash;306. doi: 10.1177/0748233710386412.\u003c/p\u003e\r\n\r\n\u003cp\u003eAntunes Dos Santos, A., M. Appel Hort, M. Culbreth, C. Lopez-Granero, M. Farina, J. B. Rocha and M. Aschner (2016). \u0026quot;Methylmercury and brain development: A review of recent literature.\u0026quot; \u003cu\u003eJ Trace Elem Med Biol\u003c/u\u003e \u003cstrong\u003e38\u003c/strong\u003e: 99-107.\u003c/p\u003e\r\n\r\n\u003cp\u003eArnold AP, Khoon ST, Rabenstein DL (1986) Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 23. Complexation of methylmercury by selenohydryl-containing amino acids and related molecules. \u003cem\u003eInorganic Chemistry 25 (14), 2433-2437.\u003c/em\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eBalazs, R. (2006). \u0026quot;Trophic effect of glutamate.\u0026quot; \u003cu\u003eCurr Top Med Chem\u003c/u\u003e \u003cstrong\u003e6\u003c/strong\u003e(10): 961-968.\u003c/p\u003e\r\n\r\n\u003cp\u003eBoron, WF and Boulpaep, EL (2005) Medical Physiology. Elsevier. Philadelphia.\u003c/p\u003e\r\n\r\n\u003cp\u003eBranco, V., J. Canario, J. Lu, A. Holmgren and C. Carvalho (2012). \u0026quot;Mercury and selenium interaction in vivo: effects on thioredoxin reductase and glutathione peroxidase.\u0026quot; Free Radic Biol Med 52(4): 781-793.\u003c/p\u003e\r\n\r\n\u003cp\u003eBranco, V., L. Coppo, S. Sola, J. Lu, C. M. P. Rodrigues, A. Holmgren and C. Carvalho (2017). \u0026quot;Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury.\u0026quot; Redox Biol 13: 278-287.\u003c/p\u003e\r\n\r\n\u003cp\u003eBridges, K., Venables, B., Roberts, A., 2017. Effects of dietary methylmercury on the dopaminergic system of adult fathead minnows and their offspring. Environ Toxicol Chem 36, 1077-1084.\u003c/p\u003e\r\n\r\n\u003cp\u003eBrown, G. C. and A. Bal-Price (2003). \u0026quot;Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria.\u0026quot; \u003cu\u003eMol Neurobiol\u003c/u\u003e \u003cstrong\u003e27\u003c/strong\u003e(3): 325-355.\u003c/p\u003e\r\n\r\n\u003cp\u003eBrzozowski, M. J., P. Jenner and S. Rose (2015). \u0026quot;Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP(+)-induced neuronal toxicity.\u0026quot; \u003cu\u003eJ Neural Transm\u003c/u\u003e \u003cstrong\u003e122\u003c/strong\u003e(6): 779-788.\u003c/p\u003e\r\n\r\n\u003cp\u003eCagiano, R., et al. (1990). \u0026quot;Evidence that exposure to methyl mercury during gestation induces behavioral and neurochemical changes in offspring of rats.\u0026quot; Neurotoxicol Teratol \u003cstrong\u003e12\u003c/strong\u003e(1): 23-28.\u003c/p\u003e\r\n\r\n\u003cp\u003eCarvalho, C.M. et al. (2008) Inhibition of the human thioredoxin system. A molecular mechanism of mercury toxicity. J Biol Chem 283, 11913-11923.\u003c/p\u003e\r\n\r\n\u003cp\u003eCarvalho, C.M.L. et al. (2011), Effects of selenite and chelating agents on mammalian thioredoxin reductase inhibited by mercury: Implications for treatment of mercury poisoning(. \u003cem\u003eFASEB Journal\u003c/em\u003e, 25 (1), pp. 370-381.\u003c/p\u003e\r\n\r\n\u003cp\u003eCastoldi, A. F., C. Johansson, N. Onishchenko, T. Coccini, E. Roda, M. Vahter, S. Ceccatelli and L. Manzo (2008a). \u0026quot;Human developmental neurotoxicity of methylmercury: impact of variables and risk modifiers.\u0026quot; \u003cu\u003eRegul Toxicol Pharmacol\u003c/u\u003e \u003cstrong\u003e51\u003c/strong\u003e(2): 201-214.\u003c/p\u003e\r\n\r\n\u003cp\u003eCastoldi, A. F., N. Onishchenko, C. Johansson, T. Coccini, E. Roda, M. Vahter, S. Ceccatelli and L. Manzo (2008b). \u0026quot;Neurodevelopmental toxicity of methylmercury: Laboratory animal data and their contribution to human risk assessment.\u0026quot; \u003cu\u003eRegul Toxicol Pharmacol\u003c/u\u003e \u003cstrong\u003e51\u003c/strong\u003e(2): 215-229.\u003c/p\u003e\r\n\r\n\u003cp\u003eCeccatelli, S., R. Bose, K. Edoff, N. Onishchenko and S. Spulber (2013). \u0026quot;Long-lasting neurotoxic effects of exposure to methylmercury during development.\u0026quot; \u003cu\u003eJ Intern Med\u003c/u\u003e \u003cstrong\u003e273\u003c/strong\u003e(5): 490-497.\u003c/p\u003e\r\n\r\n\u003cp\u003eCharleston, J. S., R. L. Body, R. P. Bolender, N. K. Mottet, M. E. Vahter and T. M. Burbacher (1996). \u0026quot;Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure.\u0026quot; \u003cu\u003eNeuroToxicology\u003c/u\u003e \u003cstrong\u003e17\u003c/strong\u003e: 127-138.\u003c/p\u003e\r\n\r\n\u003cp\u003eChao, C. C., S. Hu and P. K. Peterson (1995). \u0026quot;Glia, cytokines, and neurotoxicity.\u0026quot; \u003cu\u003eCrit.Rev.Neurobiol.\u003c/u\u003e \u003cstrong\u003e9\u003c/strong\u003e: 189-205.\u003c/p\u003e\r\n\r\n\u003cp\u003eCosta LG, Aschner M, Vitalone A, Syversen T, Soldin OP (2004) Developmental neuropathology of environmental agents. Annu Rev Pharmacol Toxicol 44:87-110\u003c/p\u003e\r\n\r\n\u003cp\u003eCoyle, J. and Puttfarcken, P. (1993) \u0026lsquo;Glutamate Toxicity\u0026rsquo;, \u003cem\u003eScience\u003c/em\u003e, 262, pp. 689\u0026ndash;95.\u003c/p\u003e\r\n\r\n\u003cp\u003eCurtis, J. T., A. N. Hood, Y. Chen, G. P. Cobb and D. R. Wallace (2010). \u0026quot;Chronic metals ingestion by prairie voles produces sex-specific deficits in social behavior: an animal model of autism.\u0026quot; \u003cu\u003eBehav Brain Res\u003c/u\u003e \u003cstrong\u003e213\u003c/strong\u003e(1): 42-49.\u003c/p\u003e\r\n\r\n\u003cp\u003eDebes, F., E. Budtz-Jorgensen, P. Weihe, R. F. White and P. Grandjean (2006). \u0026quot;Impact of prenatal methylmercury exposure on neurobehavioral function at age 14 years.\u0026quot; \u003cu\u003eNeurotoxicol Teratol\u003c/u\u003e \u003cstrong\u003e28\u003c/strong\u003e(5): 536-547.\u003c/p\u003e\r\n\r\n\u003cp\u003eDekkers, M.P., Nikoletopoulou, V., Barde, Y.A., 2013. Cell biology in neuroscience: Death of developing neurons: new insights and implications for connectivity. J Cell Biol 203, 385-393.\u003c/p\u003e\r\n\r\n\u003cp\u003eD\u0026#39;Hooge R, De Deyn PP. (2001). Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev. 36: 60-90.\u003c/p\u003e\r\n\r\n\u003cp\u003eDommergues, M. A., F. Plaisant, C. Verney and P. Gressens (2003). \u0026quot;Early microglial activation following neonatal excitotoxic brain damage in mice: a potential target for neuroprotection.\u0026quot; \u003cu\u003eNeuroscience\u003c/u\u003e \u003cstrong\u003e121\u003c/strong\u003e(3): 619-628.\u003c/p\u003e\r\n\r\n\u003cp\u003eEddins, D., et al. (2008). \u0026quot;Mercury-induced cognitive impairment in metallothionein-1/2 null mice.\u0026quot; Neurotoxicol Teratol \u003cstrong\u003e30\u003c/strong\u003e(2): 88-95.\u003c/p\u003e\r\n\r\n\u003cp\u003eEskes, C., P. Honegger, L. Juillerat-Jeanneret and F. Monnet-Tschudi (2002). \u0026quot;Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release.\u0026quot; \u003cu\u003eGlia\u003c/u\u003e \u003cstrong\u003e37\u003c/strong\u003e(1): 43-52.\u003c/p\u003e\r\n\r\n\u003cp\u003eFalluel-Morel, A., Sokolowski, K., Sisti, H.M., Zhou, X., Shors, T.J., Dicicco-Bloom, E., 2007. Developmental mercury exposure elicits acute hippocampal cell death, reductions in neurogenesis, and severe learning deficits during puberty. J Neurochem 103, 1968-1981.\u003c/p\u003e\r\n\r\n\u003cp\u003eFarina, M., J. B. Rocha and M. Aschner (2011). \u0026quot;Mechanisms of methylmercury-induced neurotoxicity: evidence from experimental studies.\u0026quot; \u003cu\u003eLife Sci\u003c/u\u003e \u003cstrong\u003e89\u003c/strong\u003e(15-16): 555-563.\u003c/p\u003e\r\n\r\n\u003cp\u003eFerraro, L., Tomasini, M.C., Tanganelli, S., Mazza, R., Coluccia, A., Carratu, M.R., Gaetani, S., Cuomo, V., Antonelli, T., 2009. Developmental exposure to methylmercury elicits early cell death in the cerebral cortex and long-term memory deficits in the rat. Int J Dev Neurosci 27, 165-174.\u003c/p\u003e\r\n\r\n\u003cp\u003eFeatherstone, D. E. (2010). \u0026quot;Intercellular glutamate signaling in the nervous system and beyond.\u0026quot; \u003cu\u003eACS Chem Neurosci\u003c/u\u003e \u003cstrong\u003e1\u003c/strong\u003e(1): 4-12.\u003c/p\u003e\r\n\r\n\u003cp\u003eFeng, S., Xu, Z., Liu, W., Li, Y., Deng, Y., Xu, B., 2014. Preventive effects of dextromethorphan on methylmercury-induced glutamate dyshomeostasis and oxidative damage in rat cerebral cortex. Biol Trace Elem Res 159, 332-345.\u003c/p\u003e\r\n\r\n\u003cp\u003eFonfria, E., Vilaro, M.T., Babot, Z., Rodriguez-Farre, E., Sunol, C., 2005. Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cells. J Neurosci Res 79, 545-553.\u003c/p\u003e\r\n\r\n\u003cp\u003eFranco, J. L. \u003cem\u003eet al.\u003c/em\u003e (2006) \u0026lsquo;Cerebellar thiol status and motor deficit after lactational exposure to methylmercury\u0026rsquo;, \u003cem\u003eEnvironmental Research\u003c/em\u003e, 102(1), pp. 22\u0026ndash;28. doi: 10.1016/j.envres.2006.02.003.\u003c/p\u003e\r\n\r\n\u003cp\u003eFranco, J. L., T. Posser, P. R. Dunkley, P. W. Dickson, J. J. Mattos, R. Martins, A. C. Bainy, M. R. Marques, A. L. Dafre and M. Farina (2009). \u0026quot;Methylmercury neurotoxicity is associated with inhibition of the antioxidant enzyme glutathione peroxidase.\u0026quot; Free Radic Biol Med 47(4): 449-457.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003ca name=\"_ENREF_45\"\u003eFritsche, E., Crofton, K.M., Hernandez, A.F., Hougaard Bennekou, S., Leist, M., Bal-Price, A., Reaves, E., Wilks, M.F., Terron, A., Solecki, R., Sachana, M.,Gourmelon, A., 2017. OECD/EFSA workshop on developmental neurotoxicity (DNT): The use of non-animal test methods for regulatory purposes. ALTEX 34, 311-315.\u003c/a\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eFujimura, M. and F. Usuki (2017). \u0026quot;In situ different antioxidative systems contribute to the site-specific methylmercury neurotoxicity in mice.\u0026quot; Toxicology 392: 55-63.\u003c/p\u003e\r\n\r\n\u003cp\u003eGeier, M.C., Minick, D. J., Truong, L., Tilton, S., Anderson, K.A., Tanguay, R.L., 2018. Systematic developmental neurotoxicity assessment of a representative PAH Superfund mixture using zebrafish. TAAP DNT Special Issue (\u003cem\u003eSubmitted\u003c/em\u003e).\u003c/p\u003e\r\n\r\n\u003cp\u003eGlaser, V., B. Moritz, A. Schmitz, A. L. Dafre, E. M. Nazari, Y. M. Rauh Muller, L. Feksa, M. R. Straliottoa, A. F. de Bem, M. Farina, J. B. da Rocha and A. Latini (2013). \u0026quot;Protective effects of diphenyl diselenide in a mouse model of brain toxicity.\u0026quot; Chem Biol Interact 206(1): 18-26.\u003c/p\u003e\r\n\r\n\u003cp\u003eGlover, C. N., et al. (2009). \u0026quot;Methylmercury speciation influences brain gene expression and behavior in gestationally-exposed mice pups.\u0026quot; Toxicol Sci \u003cstrong\u003e110\u003c/strong\u003e(2): 389-400.\u003c/p\u003e\r\n\r\n\u003cp\u003eGrandjean P, Weihe P, White RF, et al. (1997) Cognitive deficit in 7-year-old children with prenatal exposure to methylmercury. Neurotoxicol Teratol 19(6):417-28 doi:10.1016/s0892-0362(97)00097-4\u003c/p\u003e\r\n\r\n\u003cp\u003eGrandjean P, White RF (1999) Effects of methylmercury exposure on neurodevelopment. JAMA 281:896\u003c/p\u003e\r\n\r\n\u003cp\u003eGrandjean, P. and P. J. Landrigan (2006). \u0026quot;Developmental neurotoxicity of industrial chemicals.\u0026quot; \u003cu\u003eLancet\u003c/u\u003e \u003cstrong\u003e368\u003c/strong\u003e(9553): 2167-2178.\u003c/p\u003e\r\n\r\n\u003cp\u003eGrandjean, P. and Landrigan, P. J. (2014) \u0026lsquo;Neurobehavioural effects of developmental toxicity\u0026rsquo;, \u003cem\u003eThe Lancet Neurology\u003c/em\u003e, 13(3), pp. 330\u0026ndash;338. doi: 10.1016/S1474-4422(13)70278-3.\u003c/p\u003e\r\n\r\n\u003cp\u003eHan S, Zhu M, Yuan Z, Li X (2001) A methylene blue-mediated enzyme electrode for the determination of trace mercury (II), mercury (I), methylmercury, and mercury-glutathione complex. Biosensors \u0026amp; Bioelectronics. 16\u0026nbsp;: 9-16.\u003c/p\u003e\r\n\r\n\u003cp\u003eHuang, C.F., Hsu, C.J., Liu, S.H., Lin-Shiau, S.Y., 2008. Neurotoxicological mechanism of methylmercury induced by low-dose and long-term exposure in mice: oxidative stress and down-regulated Na+/K(+)-ATPase involved. Toxicol Lett 176(3), 188-197.\u003c/p\u003e\r\n\r\n\u003cp\u003eJacob, S., Thangarajan, S., 2017. Effect of Gestational Intake of Fisetin (3,3\u0026#39;,4\u0026#39;,7-Tetrahydroxyflavone) on Developmental Methyl Mercury Neurotoxicity in F1 Generation Rats. Biol Trace Elem Res 177, 297-315.\u003c/p\u003e\r\n\r\n\u003cp\u003eJoshi, D., M. D. Kumar, S. A. Kumar and S. Sangeeta (2014). \u0026quot;Reversal of methylmercury-induced oxidative stress, lipid peroxidation, and DNA damage by the treatment of N-acetyl cysteine: a protective approach.\u0026quot; J Environ Pathol Toxicol Oncol 33(2): 167-182.\u003c/p\u003e\r\n\r\n\u003cp\u003eJuarez BI, Martinez ML, Montante M, Dufour L, Garcia E, Jimenez-Capdeville ME (2002) Methylmercury increases glutamate extracellular levels in frontal cortex of awake rats. Neurotoxicol Teratol 24(6):767-71\u003c/p\u003e\r\n\r\n\u003cp\u003eKern, J. K., D. A. Geier, L. K. Sykes, B. E. Haley and M. R. Geier (2016). \u0026quot;The relationship between mercury and autism: A comprehensive review and discussion.\u0026quot; \u003cu\u003eJ Trace Elem Med Biol\u003c/u\u003e \u003cstrong\u003e37\u003c/strong\u003e: 8-24.\u003c/p\u003e\r\n\r\n\u003cp\u003eKraft, A. D. and G. J. Harry (2011). \u0026quot;Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity.\u0026quot; \u003cu\u003eInt J Environ Res Public Health\u003c/u\u003e \u003cstrong\u003e8\u003c/strong\u003e(7): 2980-3018.\u003c/p\u003e\r\n\r\n\u003cp\u003eLakshmi, D. \u003cem\u003eet al.\u003c/em\u003e (2012) \u0026lsquo;Ameliorating effect of fish oil on acrylamide induced oxidative stress and neuronal apoptosis in cerebral cortex\u0026rsquo;, \u003cem\u003eNeurochemical Research\u003c/em\u003e, 37(9), pp. 1859\u0026ndash;1867. doi: 10.1007/s11064-012-0794-1.\u003c/p\u003e\r\n\r\n\u003cp\u003eLam, H. S., K. M. Kwok, P. H. Chan, H. K. So, A. M. Li, P. C. Ng and T. F. Fok (2013). \u0026quot;Long term neurocognitive impact of low dose prenatal methylmercury exposure in Hong Kong.\u0026quot; \u003cu\u003eEnviron Int\u003c/u\u003e \u003cstrong\u003e54\u003c/strong\u003e: 59-64.\u003c/p\u003e\r\n\r\n\u003cp\u003eLanda, R. J. (2008). \u0026quot;Diagnosis of autism spectrum disorders in the first 3 years of life.\u0026quot; \u003cu\u003eNat Clin Pract Neurol\u003c/u\u003e \u003cstrong\u003e4\u003c/strong\u003e(3): 138-147.\u003c/p\u003e\r\n\r\n\u003cp\u003eLiu, J. and S. Rozovsky (2013). \u0026quot;Contribution of selenocysteine to the peroxidase activity of selenoprotein S.\u0026quot; Biochemistry 52(33): 5514-5516.\u003c/p\u003e\r\n\r\n\u003cp\u003eLoke, Y. J., A. J. Hannan and J. M. Craig (2015). \u0026quot;The Role of Epigenetic Change in Autism Spectrum Disorders.\u0026quot; \u003cu\u003eFront Neurol\u003c/u\u003e \u003cstrong\u003e6\u003c/strong\u003e: 107.\u003c/p\u003e\r\n\r\n\u003cp\u003eLu, T. H. \u003cem\u003eet al.\u003c/em\u003e (2011) \u0026lsquo;Involvement of oxidative stress-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury\u0026rsquo;, \u003cem\u003eToxicology Letters\u003c/em\u003e. Elsevier Ireland Ltd, 204(1), pp. 71\u0026ndash;80. doi: 10.1016/j.toxlet.2011.04.013.\u003c/p\u003e\r\n\r\n\u003cp\u003eMalqui H, Anarghou H, Ouardi FZ, Ouasmi N, Najimi M, Chigr F., Continuous Exposure to Inorganic Mercury Affects Neurobehavioral and Physiological Parameters in Mice., J Mol Neurosci. 2018 Oct;66(2):291-305. doi: 10.1007/s12031-018-1176-1. Epub 2018 Sep\u003c/p\u003e\r\n\r\n\u003cp\u003eMarkesbery, W. R. (1997) \u0026lsquo;Oxidative stress hypothesis in Alzheimer\u0026rsquo;s disease\u0026rsquo;, \u003cem\u003eFree Radical Biology and Medicine\u003c/em\u003e, 23(1), pp. 134\u0026ndash;147. doi: 10.1016/S0891-5849(96)00629-6.\u003c/p\u003e\r\n\r\n\u003cp\u003eMeinerz, D. F., M. T. de Paula, B. Comparsi, M. U. Silva, A. E. Schmitz, H. C. Braga, P. S. Taube, A. L. Braga, J. B. Rocha, A. L. Dafre, M. Farina, J. L. Franco and T. Posser (2011). \u0026quot;Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions.\u0026quot; Braz J Med Biol Res 44(11): 1156-1163.\u003c/p\u003e\r\n\r\n\u003cp\u003eMonnet-Tschudi, F., M. G. Zurich and P. Honegger (1996). \u0026quot;Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon.\u0026quot; \u003cu\u003eBrain Res\u003c/u\u003e \u003cstrong\u003e741\u003c/strong\u003e(1-2): 52-59.\u003c/p\u003e\r\n\r\n\u003cp\u003eMorken, T.S., Sonnewald, U., Aschner, M., Syversen, T. (2005). Effects of methylmercury on primary brain cells in mono- and co-culture. Toxicol Sci 87, 169-175.\u003c/p\u003e\r\n\r\n\u003cp\u003eMorris, G., B. K. Puri, R. E. Frye and M. Maes (2017). \u0026quot;The Putative Role of Environmental Mercury in the Pathogenesis and Pathophysiology of Autism Spectrum Disorders and Subtypes.\u0026quot; \u003cu\u003eMol Neurobiol\u003c/u\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eMorris, R.G.M. (1981). Spatial localization does not require the presence of local cues. \u003cu\u003eLearn Motiv.\u003c/u\u003e \u003cstrong\u003e12\u003c/strong\u003e: 239-260.\u003c/p\u003e\r\n\r\n\u003cp\u003eMutter, J., J. Naumann, C. Sadaghiani, R. Schneider and H. Walach (2004). \u0026quot;Alzheimer disease: mercury as pathogenetic factor and apolipoprotein E as a moderator.\u0026quot; \u003cu\u003eNeuro Endocrinol Lett\u003c/u\u003e \u003cstrong\u003e25\u003c/strong\u003e(5): 331-339.\u003c/p\u003e\r\n\r\n\u003cp\u003eNational Research Council. 2000.\u0026nbsp;\u003cem\u003eToxicological Effects of Methylmercury\u003c/em\u003e. Washington, DC: The National Academies Press. https://doi.org/10.17226/9899.\u003c/p\u003e\r\n\r\n\u003cp\u003eOliveira, C. S., B. C. Piccoli, M. Aschner and J. B. T. Rocha (2017). \u0026quot;Chemical Speciation of Selenium and Mercury as Determinant of Their Neurotoxicity.\u0026quot; \u003cu\u003eAdv Neurobiol\u003c/u\u003e \u003cstrong\u003e18\u003c/strong\u003e: 53-83.\u003c/p\u003e\r\n\r\n\u003cp\u003eOnishchenko, N., Tamm, C., Vahter, M., Hokfelt, T., Johnson, J.A., Johnson, D.A., Ceccatelli, S., 2007. Developmental exposure to methylmercury alters learning and induces depression-like behavior in male mice. Toxicol Sci 97, 428-437.\u003c/p\u003e\r\n\r\n\u003cp\u003eOrenstein, S. T., et al. (2014). \u0026quot;Prenatal organochlorine and methylmercury exposure and memory and learning in school-age children in communities near the New Bedford Harbor Superfund site, Massachusetts.\u0026quot; \u003cu\u003eEnviron \u003c/u\u003eHealth Perspect \u003cstrong\u003e122\u003c/strong\u003e(11): 1253-1259.\u003c/p\u003e\r\n\r\n\u003cp\u003ePadilla, S., Culbreth, M., Deborah, L.H., Olin, J., Jarema, K., Jensen, K., Tennant, A., 2018. Reviewer Selection Summary - Screening for Developmental Neurotoxicity Using Larval Zebrafish: Assessing the Preparation and the Predictive Capability. TAAP DNT Special Issue (\u003cem\u003eSubmitted)\u003c/em\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003ePamies, D., P. Barreras, K. Block, G. Makri, A. Kumar, D. Wiersma, L. Smirnova, C. Zang, J. Bressler, K. M. Christian, G. Harris, G. L. Ming, C. J. Berlinicke, K. Kyro, H. Song, C. A. Pardo, T. Hartung and H. T. Hogberg (2017). \u0026quot;A human brain microphysiological system derived from induced pluripotent stem cells to study neurological diseases and toxicity.\u0026quot; \u003cu\u003eALTEX\u003c/u\u003e \u003cstrong\u003e34\u003c/strong\u003e(3): 362-376.\u003c/p\u003e\r\n\r\n\u003cp\u003ePorciuncula, L. O., J. B. Rocha, R. G. Tavares, G. Ghisleni, M. Reis and D. O. Souza (2003). \u0026quot;Methylmercury inhibits glutamate uptake by synaptic vesicles from rat brain.\u0026quot; \u003cu\u003eNeuroreport\u003c/u\u003e \u003cstrong\u003e14\u003c/strong\u003e(4): 577-580.\u003c/p\u003e\r\n\r\n\u003cp\u003eQiao Y, Huang X, Chen B, He M, Hu B 2017. In vitro study on antagonism mechanism of glutathione, sodium selenite and mercuric chloride. Talanta 171\u0026nbsp;: 262-269.\u003c/p\u003e\r\n\r\n\u003cp\u003eRoda, E., T. Coccini, D. Acerbi, A. Castoldi, G. Bernocchi and L. Manzo (2008). \u0026quot;Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: an immunohistochemical study in rat.\u0026quot; \u003cu\u003eJ Chem Neuroanat\u003c/u\u003e \u003cstrong\u003e35\u003c/strong\u003e(3): 285-294.\u003c/p\u003e\r\n\r\n\u003cp\u003eRush, T., X. Liu, A. B. Nowakowski, D. H. Petering and D. Lobner (2012). \u0026quot;Glutathione-mediated neuroprotection against methylmercury neurotoxicity in cortical culture is dependent on MRP1.\u0026quot; Neurotoxicology 33(3): 476-481.\u003c/p\u003e\r\n\r\n\u003cp\u003eRuszkiewicz, J. A., A. B. Bowman, M. Farina, J. B. T. Rocha and M. Aschner (2016). \u0026quot;Sex- and structure-specific differences in antioxidant responses to methylmercury during early development.\u0026quot; \u003cu\u003eNeurotoxicology\u003c/u\u003e \u003cstrong\u003e56\u003c/strong\u003e: 118-126.\u003c/p\u003e\r\n\r\n\u003cp\u003eSandstrom J, Broyer A, Zoia D, et al. (2017a) Potential mechanisms of development-dependent adverse effects of the herbicide paraquat in 3D rat brain cell cultures. Neurotoxicology 60:116-124 doi:10.1016/j.neuro.2017.04.010\u003c/p\u003e\r\n\r\n\u003cp\u003eSandstrom J, Eggermann E, Charvet I, et al. (2017b) Development and characterization of a human embryonic stem cell-derived 3D neural tissue model for neurotoxicity testing. Toxicol In Vitro 38:124-135 doi:10.1016/j.tiv.2016.10.001\u003c/p\u003e\r\n\r\n\u003cp\u003eSarafian, T. A. \u003cem\u003eet al.\u003c/em\u003e (1994) \u0026lsquo;Bcl-2 Expression Decreases Methyle Mercury-Induced Free-Radical Generation and Cel Killing in a Neural Cell Line\u0026rsquo;, \u003cem\u003eToxicol. Lett.\u003c/em\u003e, 74(2), pp. 149\u0026ndash;155.\u003c/p\u003e\r\n\r\n\u003cp\u003eSugiura Y, Tamai Y, Tanaka H. (1978) Selenium protection against mercury toxicity\u0026nbsp;: high binding affinity of methylmercury by selenium-containing ligands in comparison with sulfur-containing ligands. Bioinorg. Chem. 9\u0026nbsp;:167-180.\u003c/p\u003e\r\n\r\n\u003cp\u003eSokolowski, K., A. Falluel-Morel, X. Zhou and E. DiCicco-Bloom (2011). \u0026quot;Methylmercury (MeHg) elicits mitochondrial-dependent apoptosis in developing hippocampus and acts at low exposures.\u0026quot; \u003cu\u003eNeurotoxicology\u003c/u\u003e \u003cstrong\u003e32\u003c/strong\u003e(5): 535-544.\u003c/p\u003e\r\n\r\n\u003cp\u003eSokolowski, K., M. Obiorah, K. Robinson, E. McCandlish, B. Buckley and E. DiCicco-Bloom (2013). \u0026quot;Neural stem cell apoptosis after low-methylmercury exposures in postnatal hippocampus produce persistent cell loss and adolescent memory deficits.\u0026quot; \u003cu\u003eDev Neurobiol\u003c/u\u003e \u003cstrong\u003e73\u003c/strong\u003e(12): 936-949.\u003c/p\u003e\r\n\r\n\u003cp\u003eTaetzsch, T. and M. L. Block (2013). \u0026quot;Pesticides, microglial NOX2, and Parkinson\u0026#39;s disease.\u0026quot; \u003cu\u003eJ Biochem Mol Toxicol\u003c/u\u003e \u003cstrong\u003e27\u003c/strong\u003e(2): 137-149.\u003c/p\u003e\r\n\r\n\u003cp\u003eTeixeira, F.B., Fernandes, R.M., Farias-Junior, P.M., Costa, N.M., Fernandes, L.M., Santana, L.N., Silva-Junior, A.F., Silva, M.C., Maia, C.S., Lima, R.R., 2014. Evaluation of the effects of chronic intoxication with inorganic mercury on memory and motor control in rats. Int J Environ Res Public Health 11, 9171-9185.\u003c/p\u003e\r\n\r\n\u003cp\u003eTian, S.M., Ma, Y.X., Shi, J., Lou, T.Y., Liu, S.S., Li, G.Y., 2015. Acrylamide neurotoxicity on the cerebrum of weaning rats. Neural Regen Res 10, 938-943.\u003c/p\u003e\r\n\r\n\u003cp\u003eVahter, M., A. Akesson, C. Liden, S. Ceccatelli and M. Berglund (2007). \u0026quot;Gender differences in the disposition and toxicity of metals.\u0026quot; \u003cu\u003eEnviron Res\u003c/u\u003e \u003cstrong\u003e104\u003c/strong\u003e(1): 85-95.\u003c/p\u003e\r\n\r\n\u003cp\u003evan Wijngaarden E, Thurston SW, Myers GJ, et al. (2017) Methyl mercury exposure and neurodevelopmental outcomes in the Seychelles Child Development Study Main cohort at age 22 and 24years. Neurotoxicol Teratol 59:35-42 doi:10.1016/j.ntt.2016.10.011\u003c/p\u003e\r\n\r\n\u003cp\u003eVilleneuve DL, Landesmann B, Allavena P, et al. (2018) Representing the Process of Inflammation as Key Events in Adverse Outcome Pathways. Toxicol Sci 163(2):346-352 doi:10.1093/toxsci/kfy047\u003c/p\u003e\r\n\r\n\u003cp\u003eVorhees, C.V., Williams, M.T. (2006) Morris waer maze: procedures for assessing spatial and related forms of learning and memory. \u003cu\u003eNat Protoc\u003c/u\u003e \u003cstrong\u003e1(2)\u003c/strong\u003e: 848-858.\u003c/p\u003e\r\n\r\n\u003cp\u003eWagner, C., Sudati, J.H., Nogueira, C.W., Rocha, J.B.T. (2010) In vivo and in vitro inhibition of mice thioredoxin reductase by methylmercury (2010) BioMetals, 23 (6), pp. 1171-1177.\u003c/p\u003e\r\n\r\n\u003cp\u003eWiederhold JG, Cramer CJ, Daniel K, Infante I, Bourdon B, Kretzschmar R. (2010) Equilibrium mercury isotope fractionation between dissolved Hg(II) species and thiol-bound Hg. Environ Sci Technol. 44\u0026nbsp;:4191-7. Doi\u0026nbsp;: 10.1021/es100205t.\u003c/p\u003e\r\n\r\n\u003cp\u003eXu, B., Xu, Z.F., Deng, Y., Liu, W., Yang, H.B., Wei, Y.G., 2012. Protective effects of MK-801 on methylmercury-induced neuronal injury in rat cerebral cortex: involvement of oxidative stress and glutamate metabolism dysfunction. Toxicology 300, 112-120.\u003c/p\u003e\r\n\r\n\u003cp\u003eYadav, S., S. P. Gupta, G. Srivastava, P. K. Srivastava and M. P. Singh (2012). \u0026quot;Role of secondary mediators in caffeine-mediated neuroprotection in maneb- and paraquat-induced Parkinson\u0026#39;s disease phenotype in the mouse.\u0026quot; \u003cu\u003eNeurochem Res\u003c/u\u003e \u003cstrong\u003e37\u003c/strong\u003e(4): 875-884.\u003c/p\u003e\r\n\r\n\u003cp\u003eYorifuji, T., et al. (2011). \u0026quot;Long-term exposure to methylmercury and psychiatric symptoms in residents of Minamata, Japan.\u0026quot; Environ Int \u003cstrong\u003e37\u003c/strong\u003e(5): 907-913.\u003c/p\u003e\r\n\r\n\u003cp\u003eZhang, Y., V. J. Bolivar and D. A. Lawrence (2013). \u0026quot;Maternal exposure to mercury chloride during pregnancy and lactation affects the immunity and social behavior of offspring.\u0026quot; \u003cu\u003eToxicol Sci\u003c/u\u003e \u003cstrong\u003e133\u003c/strong\u003e(1): 101-111.\u003c/p\u003e\r\n","overall_assessment":"\u003cp style=\"text-align:justify\"\u003eExperimental and epidemiological evidences indicate that compared to the adult central nervous system (CNS), the developing CNS is generally more susceptible to toxicant exposure (Costa et al., 2004; Grandjean and Landrigan, 2006). Pre-natal and post-natal exposure may have long-term consequences, i.e. not detected immediately at the end of the exposure period. Such effects on visuospatial memory for example have been described on child development in communities with chronic low level mercury exposure (Castoldi et al., 2008a; Debes et al., 2006; Grandjean et al., 2014; Lam et al., 2013).\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003eThe aim of this AOP is to capture the KEs and the KERs that occur after binding to thiol- and selenol groups of proteins involved in protection against oxidative stress, the MIE, and impairment in learning and memory, the AO, which is a neurotoxicity marker belonging to the OECD regulatory tool box. The chemical initiators used for the empirical support are methylmercury and mercury chloride, and acrylamide. Data are most extensive for mercury as stressor during development; data for acrylamide are much more limited and restricted to some KEs. Chronic, low-dose prenatal MeHg exposure from maternal consumption of fish has been associated with endpoints of neurotoxicity in children, including poor performance on neurobehavioral tests, particularly on tests of attention, fine-motor function, language, visual-spatial abilities (e.g., drawing), and verbal memory (NRC, 2000). However, it is important to note that some uncertainties remain about the effects of low dose of mercury during brain development (Grandjean et al., 1999). Epidemiological studies in Seychelles on prenatal exposure through fish consumption did not evidenced adverse effects on memory when analyses were performed at 22 and 24 years (Van Wyngaarden et al., 2017), whereas similar experiments made in the Faroe Islands revealed dysfunctions in language, attention and memory at 7 years (Grandjean et al., 1997). And a clear association was observed between mercury cord blood level and memory deficit (Grandjean et al., 1997; Debes et al., 2006). Castoldi and coworkers (2008) proposed that modulating factors, such as diet, nutrition, gender, pattern of exposure and co-exposure could explain the discrepancies of these epidemiological studies. Nevertheless, there are experimental evidences showing that the neurocognitive domain, in particular dentate gyrus, hippocampus and cortex are susceptible to the neurotoxicity of mercury in the developing brain (Sokolowski et al., 2011, 2013; Ceccatelli et al., 2013); therefore, we focus on impairment in learning and memory as the AO. Some \u0026ndash;SH- or \u0026ndash;SeH-containing proteins involved in protection against oxidative stress have been demonstrated to be inhibited by MeHg either \u003cem\u003ein vitro\u003c/em\u003e or \u003cem\u003ein vivo\u003c/em\u003e, but a causal relationship has not been established between these inhibitory effects and the final pathological events (Oliveira, 2017). However, the analysis of the essentiality of the KEs and of the weight of evidence for the KERs supports a plausible mechanistic link between the MIE and the AO.\u003c/p\u003e\r\n","background":"\u003cp style=\"text-align:justify\"\u003eThis AOP was originally started in a workshop report entitled: Adverse Outcome Pathways (AOP) relevant to Neurotoxicity and published in Critical Review in Toxicol: Bal-Price, A., Crofton, K.M., Sachana, M., Shafer, T.J., Behl, M., Forsby, A., Hargreaves, A., Landesmann, B., Lein, P.J., Louisse, J., Monnet-Tschudi, F., Paini, A., Rolaki, A., Schrattenholz, A., Sunol, C., van Thriel, C., Whelan, M., Fritsche, E., 2015. Putative adverse outcome pathways relevant to neurotoxicity. Crit Rev Toxicol 45(1), 83-91.\u003c/p\u003e\r\n\r\n\u003cp style=\"text-align:justify\"\u003eThe process of inflammation is common to many tissues and can be described by several KEs, as proposed in a dedicated workshop (Villeneuve et al., 2018). Brain inflammation called Neuroinflammation can be described by the two common KEs: Tissue resident cell, activation and pro-inflammatory mediators, increased. However, Neuroinflammation is a concept accepted by the regulators and is found in the whole literature describing brain inflammation. Therefore, in accord with the external reviewers, we decided to use the KE Neuroinflammation \u0026nbsp;for building the KERs of this AOP, but we introduced in the list of the KEs the two KEs common to the inflammatory process, as proposed in Villeneuve et al., 2018.\u003c/p\u003e\r\n","user_defined_mie":"1487: Binding, Thiol/seleno-proteins involved in protection against oxidative stress","user_defined_ao":"341: Impairment, Learning and memory","oecd_project":"1.13","oecd_status_id":1,"graphical_representation_image_uid":"2018/10/02/44te9q3hmn_Diapositive1.jpeg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2025-07-02T02:28:06.000-04:00","development_strategy":"","known_modulating_factors":"\u003cdiv\u003e\r\n\u003ctable class=\"table table-bordered table-fullwidth\"\u003e\r\n\t\u003cthead\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003cth\u003eModulating Factor (MF)\u003c/th\u003e\r\n\t\t\t\u003cth\u003eInfluence or Outcome\u003c/th\u003e\r\n\t\t\t\u003cth\u003eKER(s) involved\u003c/th\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/thead\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\u003c/div\u003e\r\n","assigned_license_id":15,"handbook_id":1,"project_129":false},{"id":18,"title":"PPARα activation in utero leading to impaired fertility in males","short_name":"PPARα activation leading to impaired fertility","corresponding_author_id":109,"abstract":"\u003cp\u003eThis AOP links the activation of Peroxisome Proliferator Activated Receptor \u0026alpha; (PPAR\u0026alpha;) to the developmental/reproductive toxicity in male. The development of this AOP relies on evidence collected from rodent models and incorporates human mechanistic and epidemiological data. The PPAR\u0026alpha; is a ligand-activated transcription factor that belongs to the nuclear receptor family, which also includes the steroid and thyroid hormone receptors. The hypothesis that PPAR\u0026alpha; action is the mechanistic basis for effects on the reproductive system arises from limited experimental data indicating relationships between activation of this receptor and impairment of steroidogenesis leading to reproductive toxicity. PPARs play important roles in the metabolic regulation of lipids, of which cholesterol, in particular, being a precursor of steroid hormones, makes the link between lipid metabolism to effects on reproduction. The key events in the pathway comprise the activation of PPAR\u0026alpha;, followed by the disruption cholesterol transport in mitochondria, impairment of hormonal balance which leads to malformation of the reproductive tract in males which may lead to impaired fertility. The PPAR\u0026alpha;-initiated AOP to rodent male developmental toxicity is a first step for structuring current knowledge about a mode of action which is neither AR-mediated nor via direct steroidogenesis enzymes inhibition. In the current form the pathway lays a strong basis for linking an endocrine mode of action with an apical endpoint, a prerequisite requirement for the identification of endocrine disrupting chemicals. This AOP is complemented with a structured data collection which will serve as the basis for further quantitative development of the pathway.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2024-05-01T15:58:42.000-04:00","status_id":1,"authors":"\u003cp\u003eMalgorzata Nepelska, Elise Grignard, Sharon Munn,\u003c/p\u003e\r\n\r\n\u003cp\u003eSystems Toxicology Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Via E. Fermi 2749, I-21027 Ispra, Varese, Italy\u003c/p\u003e\r\n\r\n\u003cp\u003eCorresponding author: sharon.munn@ec.europa.eu; elise.grignard@ec.europa.eu\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003eEmpirical information on dose-response relationships between the KEs, are not available, however there are solid empirical data that would inform a computational, predictive model for reproductive toxicity via PPAR\u0026alpha; activation.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eLife Stage Applicability\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThis AOP is relevant for developing (prenatal) male.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eTaxonomic Applicability\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThe experimental support for the pathway is mainly based on the animal (rat studies). Conflicting reports comes from the studies on mouse. Studies in mice report contradictory results. Recently, studies by Furr et al revealed that fetal T production can be inhibited by exposure to a phthalates in utero (CD-1 mice), but at a higher dose level than required in rats and causing systemic effects (Furr et al. 2014). However there are some earlier reports that chronic dietary administration of phthalates produces adverse testicular effects and reduces fertility in CD-1 mice (Heindel et al. 1989)\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eSex Applicability\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThis AOP applies to males only.\u003c/p\u003e\r\n","key_event_essentiality":"\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\" style=\"border-collapse:collapse; font-size:75%\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKRs\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eEssentiality - KEs\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003elevel of confidence\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePPAR alpha, Activation \u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003ePPAR alpha activation was found to indirectly alter the expression of genes involved in cholesterol transport in mitochondria\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003every weak\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTSPO; StAR decrease\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eAlterations in the amount of cholesterol transport proteins in mitochondria impact on the levels of substrate for steroid hormones production.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eweak\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003echolesterol transport in mitochondria, reduction\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eProduction of steroid hormones depends on the availability of cholesterol to the enzymes in the mitochondrial matrix. Decreasing the amount of cholesterol inside the mitochondria will result in a diminished amount of substrate for hormone (testosterone) synthesis.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003emoderate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTestosterone synthesis, reduction\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eThe gonads are generally considered the major source of circulating androgens. Consequently, if testosterone synthesis by testes is reduced, testosterone concentrations would be expected to decrease unless there are concurrent reductions in the rate of T catabolism.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003estrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTestosterone, reduction\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMale sexual differentiation in general depends on androgens (T, dihydrotestosterone (DHT)), disturbances in the balance of this endocrine system by either endogenous or exogenous factors lead to male reproductive tract malformation.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003estrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMale reproductive tract malformations\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eAndrogens regulate masculinization of the external genitalia. Therefore any defects in androgen biosynthesis, metabolism or action during foetal development can reproductive tract malformation.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003estrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFertility, impaired \u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eImpaired fertility is the endpoint of reproductive toxicity\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003estrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\r\n\r\n\u003ctable border=\"1\" style=\"border-collapse:collapse; font-size:75%\"\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eKERs \u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eBiological plausibility\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eLevel of confidence\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd colspan=\"3\"\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eEmpirical Support\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eLevel of confidence\u003c/strong\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cstrong\u003eInconsistencies/Uncertainties\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eDose-response\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eTemporality\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eIncidence\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePPAR alpha, Activation \u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003e=\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTranslator protein (TSPO), Decrease\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eThere is functional relationship between PPAR\u0026alpha; activation and reduction in TSPO levels.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eVery Weak\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEs occur at similar dose levels\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eoccurrence of the key events at similar dose and time point\u003c/li\u003e\r\n\t\t\t\t\u003cli\u003eSupport for solid temporal relationship is lacking\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eVery Weak\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSome conflicting data\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePPAR alpha, Activation \u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003e=\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSteroidogenic acute regulatory protein (StAR), decrease\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eThere is functional relationship between PPAR\u0026alpha; activation and reduction in StAR levels.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWeak\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEs occur at similar dose levels\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eSupport for solid temporal relationship is lacking.\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eWeak\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSome conflicting data\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSteroidogenic acute regulatory protein (StAR), decrease and Translator protein (TSPO), Decrease\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003e=\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003echolesterol transport in mitochondria, reduction\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eChanges in cholesterol transport proteins can generally be assumed to directly impact levels of cholesterol transport.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eModerate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEs occur at similar dose levels\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eSupport for solid temporal relationship is lacking.\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eModerate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSome conflicting data\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003echolesterol transport in mitochondria, reduction\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003e=\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003etestosterone synthesis, reduction\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eDecreasing the amount of cholesterol inside the mitochondria (e. g by decreasing the expression of enzymes like StAR or TSOP) will result in a diminished amount of substrate for hormone (testosterone) synthesis.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eModerate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEs occur at similar dose levels\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eoccurrence of the key events at similar dose and time point\u003c/li\u003e\r\n\t\t\t\t\u003cli\u003eSupport for solid temporal relationship is lacking.\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eModerate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eSome conflicting data\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003etestosterone, reduction\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003e=\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMale reproductive tract malformations\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eReduction in testosterone (T) levels produced in the Leydig cell subsequently lowers the availability of its metabolite; Dihydrotestosterone (DHT).that regulates masculinization of external genitalia. Therefore any defects in androgen biosynthesis, metabolism or action during development can cause male reproductive tract malformation.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eStrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEs occur at similar dose levels\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eoccurrence of the key events at similar dose and time point\u003c/li\u003e\r\n\t\t\t\t\u003cli\u003eSupport for solid temporal relationship is lacking.\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eStrong\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eNo conflicting data\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMale reproductive tract malformations\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003e=\u0026gt;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\r\n\t\t\t\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFertility, impaired \u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eMale reproductive tract malformations (congenital malformation of male genitalia) comprise any physical abnormality of the male internal or external genitalia present at birth, which may impair on fertility later in life\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eModerate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eKEs occur at similar dose levels\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cul\u003e\r\n\t\t\t\t\u003cli\u003eoccurrence of the key events at similar dose and time point\u003c/li\u003e\r\n\t\t\t\u003c/ul\u003e\r\n\r\n\t\t\t\u003cp\u003eSupport for solid temporal relationship is lacking.\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eModerate\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\r\n\t\t\t\u003cp\u003eNo conflicting data\u003c/p\u003e\r\n\t\t\t\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eTable 1 Weight of Evidence Summary Table. The underlying questions for the content of the table: Dose-response Does the empirical evidence support that a change in KEup leads to an appropriate change in KEdown?; Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup \u0026gt; than that for KEdown?: Incidence Is there higher incidence of KEup than of KEdown?; Inconsistencies/Uncertainties: Are there inconsistencies in empirical support across taxa, species and stressors that don\u0026rsquo;t align with expected pattern for hypothesized AOP? n.a not applicable\u003c/p\u003e\r\n","quantitative_considerations":"\u003cp\u003eThis AOP is qualitatively described; however it contains also data that may be used for further development of quantitative description.\u003c/p\u003e\r\n","optional_considerations":"\u003cp\u003e1. The AOP describes a pathway which allows for the detection of sex steroid--related endocrine disrupting modes of action, with focus on the identification of substances which affect the reproductive system. In the current form the pathway lays a strong basis for linking endocrine mode of action with an apical endpoint, a prerequisite requirement for identification of endocrine disrupting chemicals (EDC).\u003c/p\u003e\r\n\r\n\u003cp\u003eEDCs require specific evaluation under REACH (1907/2006, Registration, Evaluation, Authorisation and Restriction of Chemicals (EU, 2006)), the revised European plant protection product regulation 1107/2009 (EU, 2009) and use of biocidal products 528/2012 EC (EU, 2012).Amongst other agencies the US EPA is also giving particular attention to EDCs (EPA, 1998).\u003c/p\u003e\r\n\r\n\u003cp\u003e2. This AOP structurally represents current knowledge of the pathway from PPAR\u0026alpha; activation to impaired fertility that may provide a basis for development (and interpretation) of strategies for Integrated Approaches to Testing Assessment (IATA) to identify similar substances that may operate via the same pathway related tosex steroids disruptionand effects on reproductive tract and fertility. This AOP forms the starting point on an AOP network mapping to modes of action for endocrine disruption.\u003c/p\u003e\r\n\r\n\u003cp\u003e3. The AOP could inform the development of quantitative structure activity relationships, read-across models, and/or systems biology models to prioritize chemicals for further testing.\u003c/p\u003e\r\n","references":"\u003cp\u003eAkingbemi, B. T. 2001. \u0026ldquo;Modulation of Rat Leydig Cell Steroidogenic Function by Di(2-Ethylhexyl)Phthalate.\u0026rdquo; Biology of Reproduction 65 (4) (October 1): 1252\u0026ndash;1259. doi:10.1095/biolreprod65.4.1252. \u003ca class=\"external free\" href=\"http://www.biolreprod.org/content/65/4/1252.long\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.biolreprod.org/content/65/4/1252.long\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eArlt, W, R J Auchus, and W L Miller. 2001. \u0026ldquo;Thiazolidinediones but Not Metformin Directly Inhibit the Steroidogenic Enzymes P450c17 and 3beta -Hydroxysteroid Dehydrogenase.\u0026rdquo; The Journal of Biological Chemistry 276 (20) (May 18): 16767\u0026ndash;71. doi:10.1074/jbc.M100040200. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/11278997\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/11278997\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eBarak, Y, M C Nelson, E S Ong, Y Z Jones, P Ruiz-Lozano, K R Chien, A Koder, and R M Evans. 1999. \u0026ldquo;PPAR Gamma Is Required for Placental, Cardiac, and Adipose Tissue Development.\u0026rdquo; Molecular Cell 4 (4) (October): 585\u0026ndash;95. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/10549290\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/10549290\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eBloomgarden, Z T, W Futterweit, and L Poretsky. 2001. \u0026ldquo;Use of Insulin-Sensitizing Agents in Patients with Polycystic Ovary Syndrome.\u0026rdquo; Endocrine Practice : Official Journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists 7 (4): 279\u0026ndash;86. doi:10.4158/EP.7.4.279. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/11497481\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/11497481\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eBoberg, Julie, Stine Metzdorff, Rasmus Wortziger, Marta Axelstad, Leon Brokken, Anne Marie Vinggaard, Majken Dalgaard, and Christine Nellemann. 2008. \u0026ldquo;Impact of Diisobutyl Phthalate and Other PPAR Agonists on Steroidogenesis and Plasma Insulin and Leptin Levels in Fetal Rats.\u0026rdquo; Toxicology 250 (2-3) (September 4): 75\u0026ndash;81. doi:10.1016/j.tox.2008.05.020. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/18602967\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/18602967\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eBorch, Julie, Stine Broeng Metzdorff, Anne Marie Vinggaard, Leon Brokken, and Majken Dalgaard. 2006. \u0026ldquo;Mechanisms Underlying the Anti-Androgenic Effects of Diethylhexyl Phthalate in Fetal Rat Testis.\u0026rdquo; Toxicology 223 (1-2) (June 1): 144\u0026ndash;55. doi:10.1016/j.tox.2006.03.015. \u003ca class=\"external free\" href=\"http://www.sciencedirect.com/science/article/pii/S0300483X0600165X\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.sciencedirect.com/science/article/pii/S0300483X0600165X\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eCorton, J Christopher, Michael L Cunningham, B Timothy Hummer, Christopher Lau, Bette Meek, Jeffrey M Peters, James A Popp, Lorenz Rhomberg, Jennifer Seed, and James E Klaunig. 2014. \u0026ldquo;Mode of Action Framework Analysis for Receptor-Mediated Toxicity: The Peroxisome Proliferator-Activated Receptor Alpha (PPAR\u0026alpha;) as a Case Study.\u0026rdquo; Critical Reviews in Toxicology 44 (1) (January): 1\u0026ndash;49. doi:10.3109/10408444.2013.835784. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/24180432\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/24180432\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eCorton, J. Christopher, and Paula J Lapinskas. 2004. \u0026ldquo;Peroxisome Proliferator-Activated Receptors: Mediators of Phthalate Ester-Induced Effects in the Male Reproductive Tract?\u0026rdquo; Toxicological Sciences 83 (1) (October 13): 4\u0026ndash;17. doi:10.1093/toxsci/kfi011. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/15496498\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/15496498\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eCouto, Jana\u0026iacute;na A, Karina L A Saraiva, Cleiton D Barros, Daniel P Udrisar, Christina A Peixoto, Juliany S B C\u0026eacute;sar Vieira, Maria C Lima, Suely L Galdino, Ivan R Pitta, and Maria I Wanderley. 2010. \u0026ldquo;Effect of Chronic Treatment with Rosiglitazone on Leydig Cell Steroidogenesis in Rats: In Vivo and Ex Vivo Studies.\u0026rdquo; Reproductive Biology and Endocrinology : RB\u0026amp;E 8 (1) (January): 13. doi:10.1186/1477-7827-8-13. \u003ca class=\"external free\" href=\"http://www.rbej.com/content/8/1/13\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.rbej.com/content/8/1/13\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eDavid, RM. 2006. \u0026ldquo;Proposed Mode of Action for in Utero Effects of Some Phthalate Esters on the Developing Male Reproductive Tract.\u0026rdquo; Toxicologic Pathology. doi:10.1080/01926230600642625. \u003ca class=\"external free\" href=\"http://tpx.sagepub.com/content/34/3/209.short\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://tpx.sagepub.com/content/34/3/209.short\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eDunaif, A, D Scott, D Finegood, B Quintana, and R Whitcomb. 1996. \u0026ldquo;The Insulin-Sensitizing Agent Troglitazone Improves Metabolic and Reproductive Abnormalities in the Polycystic Ovary Syndrome.\u0026rdquo; The Journal of Clinical Endocrinology and Metabolism 81 (9) (September): 3299\u0026ndash;306. doi:10.1210/jcem.81.9.8784087. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/8784087\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/8784087\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eEma, Makoto. 2002. \u0026ldquo;Antiandrogenic Effects of Dibutyl Phthalate and Its Metabolite, Monobutyl Phthalate, in Rats.\u0026rdquo; Congenital Anomalies 42 (4) (December): 297\u0026ndash;308. doi:10.1111/j.1741-4520.2002.tb00896.x. \u003ca class=\"external free\" href=\"http://doi.wiley.com/10.1111/j.1741-4520.2002.tb00896.x\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://doi.wiley.com/10.1111/j.1741-4520.2002.tb00896.x\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eFurr, Johnathan R, Christy S Lambright, Vickie S Wilson, Paul M Foster, and Leon E Gray. 2014. \u0026ldquo;A Short-Term in Vivo Screen Using Fetal Testosterone Production, a Key Event in the Phthalate Adverse Outcome Pathway, to Predict Disruption of Sexual Differentiation.\u0026rdquo; Toxicological Sciences : An Official Journal of the Society of Toxicology 140 (2) (August 1): 403\u0026ndash;24. doi:10.1093/toxsci/kfu081. \u003ca class=\"external free\" href=\"http://toxsci.oxfordjournals.org/content/140/2/403.full\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://toxsci.oxfordjournals.org/content/140/2/403.full\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eGasic, S, Y Bodenburg, M Nagamani, A Green, and R J Urban. 1998. \u0026ldquo;Troglitazone Inhibits Progesterone Production in Porcine Granulosa Cells.\u0026rdquo; Endocrinology 139 (12) (December): 4962\u0026ndash;6. doi:10.1210/endo.139.12.6385. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/9832434\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/9832434\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eGasic, S, M Nagamani, A Green, and R J Urban. 2001. \u0026ldquo;Troglitazone Is a Competitive Inhibitor of 3beta-Hydroxysteroid Dehydrogenase Enzyme in the Ovary.\u0026rdquo; American Journal of Obstetrics and Gynecology 184 (4) (March): 575\u0026ndash;9. doi:10.1067/mob.2001.111242. \u003ca class=\"external free\" href=\"http://www.sciencedirect.com/science/article/pii/S0002937801774340\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.sciencedirect.com/science/article/pii/S0002937801774340\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eGazouli, M. 2002. \u0026ldquo;Effect of Peroxisome Proliferators on Leydig Cell Peripheral-Type Benzodiazepine Receptor Gene Expression, Hormone-Stimulated Cholesterol Transport, and Steroidogenesis: Role of the Peroxisome Proliferator-Activator Receptor .\u0026rdquo; Endocrinology 143 (7) (July 1): 2571\u0026ndash;2583. doi:10.1210/en.143.7.2571. \u003ca class=\"external free\" href=\"http://endo.endojournals.org/content/143/7/2571\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://endo.endojournals.org/content/143/7/2571\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eHannas, Bethany R, Christy S Lambright, Johnathan Furr, Nicola Evans, Paul M D Foster, Earl L Gray, and Vickie S Wilson. 2012. \u0026ldquo;Genomic Biomarkers of Phthalate-Induced Male Reproductive Developmental Toxicity: A Targeted RT-PCR Array Approach for Defining Relative Potency.\u0026rdquo; Toxicological Sciences : An Official Journal of the Society of Toxicology 125 (2) (February): 544\u0026ndash;57. doi:10.1093/toxsci/kfr315. \u003ca class=\"external free\" href=\"http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3262859\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3262859\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eHeindel, J J, D K Gulati, R C Mounce, S R Russell, and J C Lamb. 1989. \u0026ldquo;Reproductive Toxicity of Three Phthalic Acid Esters in a Continuous Breeding Protocol.\u0026rdquo; Fundamental and Applied Toxicology : Official Journal of the Society of Toxicology 12 (3) (April): 508\u0026ndash;18. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/2731665\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/2731665\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eJohnson, Kamin J, Nicholas E Heger, and Kim Boekelheide. 2012. \u0026ldquo;Of Mice and Men (and Rats): Phthalate-Induced Fetal Testis Endocrine Disruption Is Species-Dependent.\u0026rdquo; Toxicological Sciences : An Official Journal of the Society of Toxicology 129 (2) (October): 235\u0026ndash;48. doi:10.1093/toxsci/kfs206. \u003ca class=\"external free\" href=\"http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3491958\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3491958\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eKempn\u0026aacute;, Petra, Gaby Hofer, Primus E Mullis, and Christa E Fl\u0026uuml;ck. 2007. \u0026ldquo;Pioglitazone Inhibits Androgen Production in NCI-H295R Cells by Regulating Gene Expression of CYP17 and HSD3B2.\u0026rdquo; Molecular Pharmacology 71 (3) (March): 787\u0026ndash;98. doi:10.1124/mol.106.028902. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/17138841\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/17138841\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eKuhl, Adam J, Susan M Ross, and Kevin W Gaido. 2007. \u0026ldquo;CCAAT/enhancer Binding Protein Beta, but Not Steroidogenic Factor-1, Modulates the Phthalate-Induced Dysregulation of Rat Fetal Testicular Steroidogenesis.\u0026rdquo; Endocrinology 148 (12) (December): 5851\u0026ndash;64. doi:10.1210/en.2007-0930. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/17884934\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/17884934\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eLapinskas, Paula J., Sherri Brown, Lisa M. Leesnitzer, Steven Blanchard, Cyndi Swanson, Russell C. Cattley, and J. Christopher Corton. 2005. \u0026ldquo;Role of PPAR\u0026alpha; in Mediating the Effects of Phthalates and Metabolites in the Liver.\u0026rdquo; Toxicology 207 (1): 149\u0026ndash;163. \u003ca class=\"external free\" href=\"http://www.sciencedirect.com/science/article/pii/S0300483X04005633\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.sciencedirect.com/science/article/pii/S0300483X04005633\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eLatini, Giuseppe, Egeria Scoditti, Alberto Verrotti, Claudio De Felice, and Marika Massaro. 2008. \u0026ldquo;Peroxisome Proliferator-Activated Receptors as Mediators of Phthalate-Induced Effects in the Male and Female Reproductive Tract: Epidemiological and Experimental Evidence.\u0026rdquo; PPAR Research 2008 (January): 359267. doi:10.1155/2008/359267. \u003ca class=\"external free\" href=\"http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2225463\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2225463\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eLehmann, Kim P, Suzanne Phillips, Madhabananda Sar, Paul M D Foster, and Kevin W Gaido. 2004. \u0026ldquo;Dose-Dependent Alterations in Gene Expression and Testosterone Synthesis in the Fetal Testes of Male Rats Exposed to Di (n-Butyl) Phthalate.\u0026rdquo; Toxicological Sciences : An Official Journal of the Society of Toxicology 81 (1) (September 1): 60\u0026ndash;8. doi:10.1093/toxsci/kfh169. \u003ca class=\"external free\" href=\"http://toxsci.oxfordjournals.org/content/81/1/60.abstract?ijkey=99364980d6548f969a82406deb6600873a38be36\u0026amp;keytype2=tf_ipsecsha\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://toxsci.oxfordjournals.org/content/81/1/60.abstract?ijkey=99364980d6548f969a82406deb6600873a38be36\u0026amp;keytype2=tf_ipsecsha\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eMinge, Cadence E, Rebecca L Robker, and Robert J Norman. 2008. \u0026ldquo;PPAR Gamma: Coordinating Metabolic and Immune Contributions to Female Fertility.\u0026rdquo; PPAR Research 2008 (January): 243791. doi:10.1155/2008/243791. \u003ca class=\"external free\" href=\"http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2246065\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2246065\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eMu, Y M, T Yanase, Y Nishi, N Waseda, T Oda, A Tanaka, R Takayanagi, and H Nawata. 2000. \u0026ldquo;Insulin Sensitizer, Troglitazone, Directly Inhibits Aromatase Activity in Human Ovarian Granulosa Cells.\u0026rdquo; Biochemical and Biophysical Research Communications 271 (3) (May 19): 710\u0026ndash;3. doi:10.1006/bbrc.2000.2701. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/10814527\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/10814527\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eMylchreest, Eve. 2000. \u0026ldquo;Dose-Dependent Alterations in Androgen-Regulated Male Reproductive Development in Rats Exposed to Di(n-Butyl) Phthalate during Late Gestation.\u0026rdquo; Toxicological Sciences 55 (1) (May 1): 143\u0026ndash;151. doi:10.1093/toxsci/55.1.143. \u003ca class=\"external free\" href=\"http://www.toxsci.oupjournals.org/cgi/doi/10.1093/toxsci/55.1.143\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.toxsci.oupjournals.org/cgi/doi/10.1093/toxsci/55.1.143\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eMylchreest, Eve, Russell C. Cattley, and Paul M. D. Foster. 1998. \u0026ldquo;Male Reproductive Tract Malformations in Rats Following Gestational and Lactational Exposure to Di( N -Butyl) Phthalate: An Antiandrogenic Mechanism?\u0026rdquo; Toxicological Sciences 43 (1) (May 1): 47\u0026ndash;60. doi:10.1093/toxsci/43.1.47. \u003ca class=\"external free\" href=\"http://toxsci.oxfordjournals.org/content/43/1/47.short?rss=1\u0026amp;ssource=mfc\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://toxsci.oxfordjournals.org/content/43/1/47.short?rss=1\u0026amp;ssource=mfc\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003ePeters, J M, M W Taubeneck, C L Keen, and F J Gonzalez. 1997. \u0026ldquo;Di(2-Ethylhexyl) Phthalate Induces a Functional Zinc Deficiency during Pregnancy and Teratogenesis That Is Independent of Peroxisome Proliferator-Activated Receptor-Alpha.\u0026rdquo; Teratology 56 (5) (November): 311\u0026ndash;6. doi:10.1002/(SICI)1096-9926(199711)56:5\u0026lt;311::AID-TERA4\u0026gt;3.0.CO;2-#. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/9451755\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/9451755\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eShipley, Jonathan M, and David J Waxman. 2004. \u0026ldquo;Simultaneous, Bidirectional Inhibitory Crosstalk between PPAR and STAT5b.\u0026rdquo; Toxicology and Applied Pharmacology 199 (3) (October 15): 275\u0026ndash;84. doi:10.1016/j.taap.2003.12.020. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/15364543\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/15364543\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eShultz, V. D. 2001. \u0026ldquo;Altered Gene Profiles in Fetal Rat Testes after in Utero Exposure to Di(n-Butyl) Phthalate.\u0026rdquo; Toxicological Sciences 64 (2) (December 1): 233\u0026ndash;242. doi:10.1093/toxsci/64.2.233. \u003ca class=\"external free\" href=\"http://toxsci.oxfordjournals.org/content/64/2/233.abstract?ijkey=b8af27acfe10695847a4e8a9b568882405d071ae\u0026amp;keytype2=tf_ipsecsha\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://toxsci.oxfordjournals.org/content/64/2/233.abstract?ijkey=b8af27acfe10695847a4e8a9b568882405d071ae\u0026amp;keytype2=tf_ipsecsha\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eThompson, Christopher J, Susan M Ross, Janan Hensley, Kejun Liu, Susanna C Heinze, S Stanley Young, and Kevin W Gaido. 2005. \u0026ldquo;Differential Steroidogenic Gene Expression in the Fetal Adrenal Gland versus the Testis and Rapid and Dynamic Response of the Fetal Testis to Di(n-Butyl) Phthalate.\u0026rdquo; Biology of Reproduction 73 (5) (November): 908\u0026ndash;17. doi:10.1095/biolreprod.105.042382. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/15987825\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/15987825\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eVeldhuis, Johannes D, George Zhang, and James C Garmey. 2002. \u0026ldquo;Troglitazone, an Insulin-Sensitizing Thiazolidinedione, Represses Combined Stimulation by LH and Insulin of de Novo Androgen Biosynthesis by Thecal Cells in Vitro.\u0026rdquo; The Journal of Clinical Endocrinology and Metabolism 87 (3) (March): 1129\u0026ndash;33. doi:10.1210/jcem.87.3.8308. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/11889176\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/11889176\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eVierhapper, H, P Nowotny, and W Waldh\u0026auml;usl. 2003. \u0026ldquo;Reduced Production Rates of Testosterone and Dihydrotestosterone in Healthy Men Treated with Rosiglitazone.\u0026rdquo; Metabolism: Clinical and Experimental 52 (2) (February): 230\u0026ndash;2. doi:10.1053/meta.2003.50028. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/12601638\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/12601638\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eWard, J M, J M Peters, C M Perella, and F J Gonzalez. 1998. \u0026ldquo;Receptor and Nonreceptor-Mediated Organ-Specific Toxicity of di(2-Ethylhexyl)phthalate (DEHP) in Peroxisome Proliferator-Activated Receptor Alpha-Null Mice.\u0026rdquo; Toxicologic Pathology 26 (2): 240\u0026ndash;6. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/9547862\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/9547862\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eWelsh, Michelle, Philippa T K Saunders, Mark Fisken, Hayley M Scott, Gary R Hutchison, Lee B Smith, and Richard M Sharpe. 2008. \u0026ldquo;Identification in Rats of a Programming Window for Reproductive Tract Masculinization, Disruption of Which Leads to Hypospadias and Cryptorchidism.\u0026rdquo; The Journal of Clinical Investigation 118 (4) (April): 1479\u0026ndash;90. doi:10.1172/JCI34241. \u003ca class=\"external free\" href=\"http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2267017\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2267017\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\u003c/a\u003e.\u003c/p\u003e\r\n\r\n\u003cp\u003eWood, Charles E, Micheal P Jokinen, Crystal L Johnson, Greg R Olson, Susan Hester, Michael George, Brian N Chorley, et al. 2014. \u0026ldquo;Comparative Time Course Profiles of Phthalate Stereoisomers in Mice.\u0026rdquo; Toxicological Sciences : An Official Journal of the Society of Toxicology 139 (1) (May): 21\u0026ndash;34. doi:10.1093/toxsci/kfu025. \u003ca class=\"external free\" href=\"http://www.ncbi.nlm.nih.gov/pubmed/24496636\" rel=\"nofollow\" target=\"_blank\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/24496636\u003c/a\u003e.\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003e\u003cstrong\u003eBiological plausibility, coherence, and consistency of the experimental evidence\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eIn the presented AOP it is hypothesized that the key events occur in a biologically plausible order prior to the development of adverse outcomes. The PPAR\u0026alpha; activators have been shown to alter steroidogenesis and impair reproduction [see reviews (Corton and Lapinskas 2004), (Latini et al. 2008), (David 2006)]. However, there are some conflicting reports on the involvement of PPAR\u0026alpha; as MIE of the proposed AOP (Johnson, Heger, and Boekelheide 2012), (David 2006). The biochemistry of steroidogenesis and the predominant role of the gonad in synthesis of the sex steroids are well established. Steroidogenesis is a complex process that is dependent on the availability of cholesterol in mitochondria. Perturbation of genes responsible for cholesterol transport and steroidogenic enzyme activities in the Leydig cell will lead to a decrease in testicular testosterone (T) production. As a consequence, androgen-dependent tissue differentiation/development is adversely affected. The physical manifestation of this event may be reproductive tract malformation and possibly leads to impaired fertility.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConcordance of dose-response relationships\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThis is a qualitative description of the pathway; the currently available studies provide quantitative information on dose-response relationships only partially. Experimental data are based on exposure to phthalates and indicate that key events of this pathway occur at similar dose levels. The effects of altered gene expression levels that are responsible for the cholesterol transport into the Leydig cells were shown at \u0026gt;50 mg/kg/bw, a dose at which foetal T was decreased and anatomical malformations (hypospadias) were produced (Mylchreest, Cattley, and Foster 1998), (Mylchreest 2000), (Akingbemi 2001), (Lehmann et al. 2004). Tailored experiments are required for the exploration of quantitative linkages.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eTemporal concordance among the key events and the adverse outcome\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThis AOP bridges two life stages: the AOs are results of the chemical exposure during a critical prenatal period for male development, the masculinization programming window (MPW), within which androgens must act to ensure the correct development of the male reproductive tract (Welsh et al. 2008). Therefore, the AOP focuses on the exposures within the MPW (15.5\u0026ndash;18.5 GD days in rats). The temporal relationship of exposure to gestation day has been investigated using phthalates and it has been demonstrated that the gestational timing of exposure is important for the production of the adverse effects on the male reproductive tract (reviewed in (Ema 2002)). Moreover, the temporal relationship between alterations of gene expression and changes in testosterone production has been investigated for phthalates (DBP) (Lehmann et al. 2004), (Thompson et al. 2005). Initial increases in gene expression are followed by decreases in the expression of genes which are associated with steroidogenesis. The observed decreased steroidogenesis and subsequent decrease in testosterone levels is well established as precursors to anatomical changes in the developing male reproductive tract. Thus, those key events of gene expression are temporally consistent with subsequent events, however complete temporal concordance studies are missing.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n\u003cstrong\u003eStrength, consistency, and specificity of association of adverse effect and initiating event\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThe strength of the chosen chemical initiators as PPAR\u0026alpha; activators was shown to partially correlate with their ability to act as a male reproductive toxicant (Corton and Lapinskas 2004). The presented key events leading to a decrease in steroidogenesis are plausible and consistent with the observed effects. There is coherence between decreased testosterone synthesis and malformations.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eAlternative mechanism(s) or MIE(s) described which may contribute/synergise the postulated AOP\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThe inhibitory effect of PPAR\u0026alpha; activation seems to be attributable to an impairment of the multistep process of cholesterol mobilization, transport into mitochondria, and steroidogenesis leading to impaired androgens production. Therefore, it is plausible that several other mechanisms may contribute to/synergise with this AOP. For example, activation of other isoforms of PPARs (PPAR\u0026beta;/\u0026delta; or/and \u0026gamma;) is hypothesised to be relevant for the pathway (Lapinskas et al. 2005), (Shipley and Waxman 2004).\u003c/p\u003e\r\n\r\n\u003cp\u003ePPAR\u0026gamma; activation\u003c/p\u003e\r\n\r\n\u003cp\u003eOpposing effects of PPAR\u0026gamma; ligands (thiazolidinediones, TZD) on androgen levels and/or production in male humans (Dunaif et al. 1996), (Bloomgarden, Futterweit, and Poretsky 2001), (Vierhapper, Nowotny, and Waldh\u0026auml;usl 2003) and animal models have been described (Kempn\u0026aacute; et al. 2007), (Gasic et al. 1998), (Mu et al. 2000), (Arlt, Auchus, and Miller 2001), (Minge, Robker, and Norman 2008), (Gasic et al. 2001), (Veldhuis, Zhang, and Garmey 2002). In rats no effects of PPAR\u0026gamma; ligand (rosiglitazone) on production or total circulating testosterone levels were seen (Boberg et al. 2008), however a decrease in basal or induced testosterone production occurred in the Leydig cells of rosiglitazone-treated rats (Couto et al. 2010).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nMoreover, there are contradicting reports as to the presence of PPAR\u0026gamma; in the foetal testes (Hannas et al. 2012). Few others transcription factors involved in regulation of lipid metabolism are hypothesized to mediate effects on fetal Leydig cell gene expression like sterol regulatory element\u0026ndash;binding protein (SREBP) (Lehmann et al. 2004), (Shultz 2001), CCAAT/enhancer-binding protein-\u0026beta; (CEBPB) (Kuhl, Ross, and Gaido 2007) or NR5A1 (also known as steroidogenic factor 1; Sf1) (Borch et al. 2006). The downstream effects in the pathway might be due to the constellation of earlier events in fetal Leydig cells leading to decrease testosterone production and connected adverse outcomes. Alternative/synergistic MIEs relating to this pathway are hypothesised in the KER description. At present there are no strong views on the other possible MIEs.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eUncertainties, inconsistencies and data gaps\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003eThe major uncertainty in this AOP is the functional relationship between (MIE) PPAR\u0026alpha; activation leading to cholesterol transport reduction; possible mechanisms have been proposed but strong experimental support is missing and some conflicting data are reported. The dose response data to support this relationship are lacking. Studies exploring the role of PPAR\u0026alpha; using PPAR\u0026alpha; knockout mice showed that prenatal exposure to phthalates caused developmental malformations in both wild-type and PPAR\u0026alpha; knockout mice, thus suggesting a PPAR\u0026alpha;-independent mechanism. However, it is difficult to draw any conclusion on the role of PPAR\u0026alpha; in phthalate-related reproductive toxicity since the intrauterine administration of phthalate (DEHP) occurred before the critical period of reproductive tract differentiation (Peters et al. 1997). Intrauterine DEHP-treated PPAR\u0026alpha;-deficient mice, developed delayed testicular, renal and developmental toxicities, but no liver toxicity, compared to wild types, thus confirming the early observation by Lee et al. about the PPAR\u0026alpha; dependence of liver response and, more importantly, indicating that DEHP may induce reproductive toxicity through both PPAR\u0026alpha;-dependent and -independent mechanism (Ward et al. 1998). PPAR\u0026alpha;-independent reproductive toxicity observed by Ward et al. may conceivably be mediated by other PPAR isoforms, such as PPAR\u0026beta; and PPAR\u0026gamma;, or by a non-receptor-mediated organ-specific mechanism (Barak et al. 1999). Other studies showed that the administration of DEHP resulted in milder testis lesions and higher testosterone levels in PPAR\u0026alpha;-null mice than in wild-type mice (Gazouli 2002). A more recent report, investigating the role of PPAR\u0026alpha;, showed decreased testosterone levels in PPAR\u0026alpha;(\u0026minus;/\u0026minus;) null control mice, suggesting a positive constitutive role for PPAR\u0026alpha; in maintaining Leydig cell steroid formation (Borch et al. 2006).\u003c/p\u003e\r\n\r\n\u003cp\u003eInconsistencies Genomic studies by Hannas et al., demonstrated that PPAR\u0026alpha; agonist Wy-14,643, did not reduce foetal testicular testosterone production following gestational day 14\u0026ndash;18 exposure, suggesting that the antiandrogenic activity of phthalates is not PPAR\u0026alpha; mediated (Hannas et al. 2012). Similarly, recent report by Furr et al. did not observe testosterone decrease after administration of Wy-14,643 in rat ( ex vivo) (Furr et al. 2014).\u003c/p\u003e\r\n\r\n\u003cp\u003eData Gaps: Complete/pathway driven studies to investigate the effects of PPARs and their role in male reproductive development are lacking. For establishing a solid quantitative and temporal coherent linkage, mode of action framework analysis for PPAR \u0026alpha; mediated developmental toxicity are needed. This approach has been applied for the involvement of PPAR \u0026alpha; in liver toxicity (Corton et al. 2014), (Wood et al. 2014).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","background":"","user_defined_mie":"227: Activation, PPARα","user_defined_ao":"406: impaired, Fertility and 348: Malformation, Male reproductive tract","oecd_project":"1.21","oecd_status_id":3,"graphical_representation_image_uid":"2016/12/02/xffqcqey_750732_PPAR__activation_leading_to_decreased_fertility.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2024-05-01T15:58:42.000-04:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":16,"handbook_id":1,"project_129":false},{"id":19,"title":"Androgen receptor antagonism leading to adverse effects in the male foetus (mammals)","short_name":"AR antagonism leading to foetal feminisation","corresponding_author_id":241,"abstract":"\u003cp\u003eThis adverse outcome pathway details the linkage between the antagonism of the androgen receptor (AR) leading to adverse effects in the male foetus. For a more detailed explanation of this pathway, with supporting references, please refer to the project report to the OECD \u003csup\u003e\u003ca href=\"#cite_note-1\"\u003e[1]\u003c/a\u003e\u003c/sup\u003e. The AR is involved in the mediation of various cellular processes including proliferation, differentiation and apoptosis in many tissues. The two main events regulated by AR mediated gene expression are urogenital tract differentiation during gestation and sexual changes during puberty. The AR can be activated by the binding of the endogenous androgens testosterone and its metabolite 5-alpha-dihydrotestosterone (DHT), which can activate gene expression at the transcription level. In mammals, virilisation of the external genitalia is driven by DHT while the differentiation of the Wolffian duct is driven by testosterone. Chemicals which bind to the AR may cause disruption by agonism, antagonism or by both mechanisms. Agonists will mimic the action of the endogenous androgens, whilst antagonists will block the receptor and prevent activation. Androgen receptor antagonists divide into steroid-like and non-steroidal compounds. Several classes or chemical categories are indicated by the data \u003csup\u003e\u003ca href=\"#cite_note-2\"\u003e[2]\u003c/a\u003e\u003c/sup\u003e\u003csup\u003e\u003ca href=\"#cite_note-3\"\u003e[3]\u003c/a\u003e\u003c/sup\u003e. These include the steroidal class (cyproterone acetate), the flutamide/ \u0026ldquo;aryl amide\u0026rdquo; class which includes bicalutamide, linuron and hydantoin analogs (such as nilutamide, vinclozin), the quinoline analog class, and the phthalimide derivatives. The best characterised class are synthetic anilides for which the model compound is flutamide.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nFlutamide exhibits potent anti-androgenic activity and in animals shows dose dependent decreases in the weight of accessory sex organs at doses of 1mg/kg and above. In utero exposure to flutamide in rats has been shown to cause feminisation of external genitalia, nipple retention and alteration of androgen-dependent testicular descent in male foetuses. A number of flutamide derivatives with in vitro binding data have demonstrated in vivo activity \u003csup\u003e\u003ca href=\"#cite_note-4\"\u003e[4]\u003c/a\u003e\u003c/sup\u003e\u003csup\u003e\u003ca href=\"#cite_note-5\"\u003e[5]\u003c/a\u003e\u003c/sup\u003e\u003csup\u003e\u003ca href=\"#cite_note-6\"\u003e[6]\u003c/a\u003e\u003c/sup\u003e\u003csup\u003e\u003ca href=\"#cite_note-7\"\u003e[7]\u003c/a\u003e\u003c/sup\u003e. In vitro the relative binding affinity (RBA) to the AR can be measured using assays which compare the competitive binding versus a control compound such as DHT or a synthetic androgen (metribolone (R1881) or mibolerone). Although this assay can measure binding it cannot distinguish between agonists and antagonists \u003csup\u003e\u003ca href=\"#cite_note-8\"\u003e[8]\u003c/a\u003e\u003c/sup\u003e\u003csup\u003e\u003ca href=\"#cite_note-9\"\u003e[9]\u003c/a\u003e\u003c/sup\u003e. Transcriptional activation in cells transfected with human AR can be used to identify agonism or antagonism with respect to that induced by a known concentration of DHT \u003csup\u003e\u003ca href=\"#cite_note-10\"\u003e[10]\u003c/a\u003e\u003c/sup\u003e. Short term in vivo studies may use the Hershberger assay or acute studies involving castrated rat models \u003csup\u003e\u003ca href=\"#cite_note-11\"\u003e[11]\u003c/a\u003e\u003c/sup\u003e.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2024-11-18T16:22:06.000-05:00","status_id":3,"authors":"","applicability_of_the_aop":"","key_event_essentiality":"","weight_of_evidence_summary":"","quantitative_considerations":"","optional_considerations":"","references":"\u003col\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-1\"\u003e\u0026uarr;\u003c/a\u003e Project report: Developmental Toxicity Associated with the Androgen Receptor Antagonism Adverse Outcome Pathway. OECD QSAR Toolbox Report, Deliverable D6.5, 2011.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-2\"\u003e\u0026uarr;\u003c/a\u003e Singh, S.M., Gauthier, S., Labrie, F., Current Medicinal Chemistry, 2000 (7) 211-247.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-3\"\u003e\u0026uarr;\u003c/a\u003e Gao, W., Bohl, C.E., Dalton, J.T., Chemical Reviews, 2005 (105) 3352-3370.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-4\"\u003e\u0026uarr;\u003c/a\u003e Morris, J.J., Hughes, L.R., Glen, A.T., Taylor, P. J., Journal of Medicinal Chemistry, 1991 (34) 447-455.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-5\"\u003e\u0026uarr;\u003c/a\u003e Singh, S.M., Gauthier, S., Labrie, F., Current Medicinal Chemistry, 2000 (7) 211-247.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-6\"\u003e\u0026uarr;\u003c/a\u003e Yin, D., He, Y., Perera, M.A., Hong, S.S., Marhefka, C., Stourman, N., Kirkovsky, L., Miller, D.D., Dalton, J.T., Molecular Pharmacology, 2003 (63) 211-223.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-7\"\u003e\u0026uarr;\u003c/a\u003e Gao, W., Bohl, C.E., Dalton, J.T., Chemical Reviews, 2005 (105) 3352-3370.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-8\"\u003e\u0026uarr;\u003c/a\u003e Singh, S.M., Gauthier, S., Labrie, F., Current Medicinal Chemistry, 2000 (7) 211-247.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-9\"\u003e\u0026uarr;\u003c/a\u003e Yin, D., He, Y., Perera, M.A., Hong, S.S., Marhefka, C., Stourman, N., Kirkovsky, L., Miller, D.D., Dalton, J.T., Molecular Pharmacology, 2003 (63) 211-223.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-10\"\u003e\u0026uarr;\u003c/a\u003e Yin, D., He, Y., Perera, M.A., Hong, S.S., Marhefka, C., Stourman, N., Kirkovsky, L., Miller, D.D., Dalton, J.T., Molecular Pharmacology, 2003 (63) 211-223.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-11\"\u003e\u0026uarr;\u003c/a\u003e Lambright, C., Ostby, J., Bobseine, K., Wilson, V., Hotchkiss, A.K., Mann, P.C., Gray, L.E., Toxicological Sciences, 2000 (56) 389-399.\u003c/li\u003e\r\n\u003c/ol\u003e\r\n","overall_assessment":"","background":"","user_defined_mie":"27: N/A, Androgen receptor, Antagonism","user_defined_ao":"337: N/A, Impairment of reproductive capacity","oecd_project":"","oecd_status_id":null,"graphical_representation_image_uid":"2016/11/29/cc5833877_AR_antagonism_leading_to_foetal_feminisation.jpg","saaop_status_id":3,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2024-11-18T16:22:06.000-05:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":17,"handbook_id":1,"project_129":false},{"id":21,"title":"Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2","short_name":"AhR mediated mortality, via COX-2","corresponding_author_id":220,"abstract":"\u003cp\u003eThis adverse outcome pathway details the linkage between activation of the aryl hydrocarbon receptor (AhR) and early life stage mortality in oviparous vertebrates.\u0026nbsp;This AOP can be initiated by a range of planar aromatic hydrocarbons, but is best known as the target of dioxin-like compounds (DLCs). These planar compounds are able to bind to the AhR causing\u0026nbsp;heterodimerization with the aryl hydrocarbon nuclear translocator (ARNT) and interaction with dioxin-responsive elements on the DNA causing an up-regulation in dioxin responsive genes. Hundreds to thousands of genes are regulated, either directly or indirectly, by the AhR. One dioxin-responsive gene is cyclooxygenase 2 (COX-2) which has roles in development of the cardiovascular system. Up-regulation in expression of COX-2 causes alteration in cardiovascular development and function which results in reduced heart pumping efficiency, reduced blood flow, and eventual cardiac collapse and death. Comparable apical manifestations of activation of the AhR have been recorded across freshwater and marine teleost and non-teleost fishes, as well as birds. Therefore, this AOP might be broadly applicable across oviparous vertebrate taxa. Despite conservation in\u0026nbsp;the AOP across taxa, great differences in sensitivity to perturbation exist both among and within taxonomic groups. Therefore, this AOP has utility in support of application toward the mechanistic understanding of adverse effects of chemicals that act as agonists of the AhR, particularly with regard to cross-chemical, cross-species, and cross-taxa extrapolation.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eIn general, biological plausibility of this AOP is strong based\u0026nbsp;heavily on evidence collected from zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) through mechanistic investigations by use of targeted knockdown of AhR, ARNT, or COX-2 and through use of selective agonists and antagonists of COX-2. Quantitative understanding is largely limited to the indirect KER between\u0026nbsp;AhR activation and early life stage mortality.\u003c/p\u003e\r\n\r\n\u003cp\u003eActivation of the AhR causes pleotropic responses, including interaction with multiple potential target genes such as CYP1A, Sox9b, and HIF1a/VEGF. Therefore, it is a challenge to elucidate the precise series of key events which link activation of the AhR to early life stage mortality. Because of this uncertainty, other AOPs, such as through the HIF1a/VEGF signalling pathway (\u003ca href=\"https://aopwiki.org/aops/150\"\u003eAOP 150\u003c/a\u003e), have also been developed. These other AOPs likely occur simultaneously with COX-2 to cause altered cardiovascular development and function leading to early life stage mortality.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":1,"authors":"\u003cp\u003eJon Doering, Ph.D., National Research Council,\u0026nbsp;US EPA Mid-Continent Ecology Division, Duluth, MN, USA, (doering.jonathon[at]epa.gov)\u003c/p\u003e\r\n\r\n\u003cp\u003eProf. Markus Hecker, Ph.D. University of Saskatchewan, Saskatoon, Saskatchewan, Canada, (markus.hecker[at]usask.ca)\u003c/p\u003e\r\n\r\n\u003cp\u003eDan Villeneuve, Ph.D., US EPA Mid-Continent Ecology Division, Duluth, MN, USA (villeneuve.dan[at]epa.gov)\u003c/p\u003e\r\n\r\n\u003cp\u003eProf. Xiaowei Zhang, Ph.D., Nanjing University, School of the Environment, Nanjing, China (Zhangxw[at]nju.edu.cn)\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003e\u003cstrong\u003eSex: \u003c/strong\u003eThis AOP is only applicable to early life stages prior to sexual differentiation.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eLife stages: \u003c/strong\u003eThis AOP is only applicable starting from embryonic development. In zebrafish, this critical window extends from fertilization to approximately 24 hours post fertilization (hpf) (Belair et al 2001; Goldstone \u0026amp; Stegeman 2008).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eTaxonomic:\u003c/strong\u003e The specific characteristics of altered cardiovascular development and function vary to some degree among taxonomic groups of vertebrates.\u003c/p\u003e\r\n\r\n\u003cp\u003eThis AOP is applicable to:\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eAll teleost and non-teleost fishes that have been investigated as embryos so far (Buckler et al 2015; Doering et al 2013; Elonen et al 1998; Johnson et al 1998; Park et al 2014; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Yamauchi et al 2006; Zabel et al 1995).\u003c/li\u003e\r\n\t\u003cli\u003eAll birds (Canga et al 1993; Cohen-Barnhouse et al 2011; Fujisawa et al 2014; Heid et al 2001; Ivnitski et al 2001; Walker \u0026amp; Catron 2000). However, some details of the AOP might be different in birds as cyclooxygenase-2 (COX-2) is believed to be up-regulated through non-genomic mechanisms in these taxa based on investigations in chicken (\u003cem\u003eGallus gallus\u003c/em\u003e) (Fujisawa et al 2014).\u003c/li\u003e\r\n\t\u003cli\u003eAmphibians and reptiles have insufficient mechanistic and early life stage mortality information to demonstrate applicability at this time. However, amphibians and reptiles express AhRs that are activated by agonists in a manner consistent with other vertebrates and express AhRs during embryonic development (Lavine et al 2005; Shoots et al 2015; Ohi et al 2003; Oka et al 2016). However, altered cardiovascular development and function and early life stage mortality have not been observed at any investigated concentration of DLC in amphibians studied to date (Jung et al 1997). This tolerance is believed to result from AhRs of amphibians having very low affinity for agonists (Lavine et al 2005; Shoots et al 2015). Therefore, it is acknowledged that this AOP is likely to be applicable to reptiles. However, it might not be applicable to amphibians due to their extreme tolerance to activation of the AhR.\u003c/li\u003e\r\n\t\u003cli\u003eCartilaginous fishes (Chondrichthyes) have insufficient mechanistic and early life stage mortality information to demonstrate applicability at this time. However, sharks and rays are known to express AhRs that are structurally comparable to AhRs of teleost fishes (Hahn 2002). Sharks and rays have also been shown to respond to exposure to agonists of the AhR through responses that are comparable to teleost fishes, specifically through induction of CYP1A (Hahn et al 1998). Therefore, it is acknowledged that this AOP is likely to be applicable to Chondrichthyes.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003eThis AOP is not applicable to:\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eMammals because the cause of mortality of the young is primarily a result of wasting syndrome and not necessarily altered cardiovascular development and function (Kopf \u0026amp; Walker 2009). Further, studies of CYP1A1 and CYP1A2 null mice (\u003cem\u003eMus musculus\u003c/em\u003e) demonstrate that wasting syndrome and mortality are mediated by CYP1A1 in mammals (Uno et al 2004).\u003c/li\u003e\r\n\t\u003cli\u003eInvertebrates because AhRs of invertebrates have less diverse functionalities relative to vertebrates, AhRs of most invertebrates likely do not\u0026nbsp;bind agonists that represent anthropogenic pollutants, and no AhR-mediated, critical adverse effects are known in invertebrates as a result of exposure to AhR agonists\u0026nbsp;(Hahn 2002; Hahn et al 1994).\u003c/li\u003e\r\n\t\u003cli\u003eJawless fishes, such as lamprey (Petromyzontiformes) and hagfish (Myxiniformes), because of a lack of measurable AhR-mediated responses (Hahn et al 1998). Although additional information is necessary for this taxa, it is currently acknowledged that this AOP is likely not applicable to jawless fishes.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","key_event_essentiality":"\u003cp\u003eSupport for essentiality for key events in the AOP was provided by a series of knockdown and targeted agonist and antagonist experiments. These investigations were conducted mainly with zebrafish as the model species and TCDD as the model agonist of the AhR.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;Rationale for essentiality calls:\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eAhR, activation: [Strong] Knockdown of AhR prevents TCDD induced alteration in cardiovascular development and function (Clark et al 2010; Hanno et al 2010; Karchner et al 1999; Prasch et al 2003; Van Tiem \u0026amp; Di Giulio 2011).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eAhR/ARNT, dimerization: [Strong] Knockdown of ARNT prevents TCDD induced alteration in cardiovascular development and function (Antkiewicz et al 2006; Prasch et al 2004). Depletion of ARNT lessens or prevents TCDD induced alteration in cardiovascular development and function (Prasch et al 2004).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eCOX-2, increase: [Strong] Knockdown of COX-2 and selective antagonists of COX-2 prevent TCDD induced alteration in cardiovascular development and function (Dong et al 2010; Teraoka et al 2008; 2014). COX-2 inducers that are not agonists of the AhR cause altered cardiovascular development and function that is consistent with activation of the AhR (Huang et al 2007). Knockdown of and selective antagonists of thromboxane A synthase 1 (CYP5A), which is down-stream of COX-2 in the prostaglandin synthesis pathway, prevents TCDD induced alteration in cardiovascular development and function (Teraoka et al 2008). Exposure to the substrate for COX-2, arachidonic acid, causes an up-regulation in COX-2 and altered cardiovascular development and function that is consistent with exposure to TCDD (Dong et al 2010).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eCardiovascular development and function, altered: [Strong] Isosmotic rearing solution prevents yolk sac edema, but has no effect on TCDD induced alteration in cardiovascular development and function or mortality (Hill et al 2004). This indicates that mortality is not caused by yolk sac edema. Knockdown of cytochrome P450 1A (CYP1A) or injection with antioxidants decreases oxidative stress but has no effect on TCDD induced alteration in cardiovascular development and function or mortality (Carney et al 2004; Scott et al 2011). This is suggestive that mortality is not caused by oxidative stress. Exposure to agonists of the AhR post-heart development lessens or prevents alteration in cardiovascular development, decreased blood flow, and cardiac failure (Carney et al 2004; Lanham et al 2012). Exposure to agonists of the AhR post-heart development dramatically reduces mortality (Carney et al 2004; Lanham et al 2012). This suggests that mortality is caused by circulatory failure as a result of cardiovascular teratogenenesis. Concentrations of DLCs tested in amphibians studied to date were not sufficient to cause altered cardiovascular development or function and no increase in mortality was observed (Jung et al 1997).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003e\u003cstrong\u003eBiological Plausibility: \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eIn general, the biological plausibility and coherence linking activation of the AhR through early life stage mortality from COX-2 induced alteration in cardiovascular development and function is strong.\u003c/li\u003e\r\n\t\u003cli\u003eThe AhR is known to have critical roles in development of the heart and therefore dysregulation of these roles would be expected to result in altered cardiac development.\u003c/li\u003e\r\n\t\u003cli\u003eThe prostaglandin synthesis pathway, of which COX-2 is a rate limiting step, is known to have roles in development of the heart (Delgado et al 2004; Gullestad \u0026amp; Aukrust 2005; Hocherl et al 2002; Huang et al 2007; Wong et al 1998;\u0026nbsp;Dong et al 2010; Huang et al 2007; Teraoka et al 2008; 2014).\u003c/li\u003e\r\n\t\u003cli\u003eA properly functioning circulatory system is widely acknowledged to be crucial for survival of vertebrates (Kardong 2006). General dysfunction of the heart or associated vasculature is widely documented to have the potential to result in mortality through cardiac failure, regardless of the mechanism of dysfunction.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConcordance of dose-response relationships: \u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThere is significant evidence showing concordance of dose-response for incidence and severity of alteration in cardiovascular development and function and subsequently mortality across at least 16 different species of fish (Buckler et al 2015; Elonen et al 1998; Huang et al 2012; Johnson et al 1998; Park et al 2014; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Yamauchi et al 2006; Zabel et al 1995) and 8 different species of birds (Cohen-Barnhouse et al 2011; Brunstrom 1990; Brunstrom \u0026amp; Andersson 1988; Hoffman et al 1996; 1998; Powell et al 1998) for several PCDDs, PCDFs, planar PCBs, and PAHs. Concordance of dose-response has not been observed in amphibians studied to date because no elevated mortality or altered cardiovascular development and function was observed at any tested concentration of agonist (Jung et al 1997).\u003c/li\u003e\r\n\t\u003cli\u003eLess is known regarding concordance of dose-response relationships for COX-2. In Japanese medaka (\u003cem\u003eOryzias latipes\u003c/em\u003e), abundance of transcript of COX-2 is significantly greater than controls at concentrations of TCDD of 0.2 ppb and greater (Dong et al 2010). Likewise, incidence of cardiovascular development and heart area were both significantly different than controls at concentrations of TCDD of 0.2 ppb and greater (Dong et al 2010).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eTemporal concordance among the key events and adverse effect:\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eAlterations in cardiovascular development or function is first observable in zebrafish around 48 hours post fertilization (hpf), while mortality does not begin to occur until around 86 hpf (Goldstone \u0026amp; Stegeman 2006).\u003c/li\u003e\r\n\t\u003cli\u003eAhR transcript and protein is first detectable in zebrafish around 24 hpf (Tanguay et al 1999).\u003c/li\u003e\r\n\t\u003cli\u003eCOX-2 transcript is first detectable in zebrafish around 6 hpf (Teraoka et al 2008). Expression of COX-2 was investigated in zebrafish exposed to TCDD at 55 and 72 hpf (Teraoka et al 2014). At 55 hpf there was a trend towards up-regulation of COX-2 (~ 1.5-fold), while at 72 hpf there was a significant up-regulation of COX-2 (~ 4.5-fold) (Teraoka et al 2014).\u003c/li\u003e\r\n\t\u003cli\u003eTherefore, there is a general temporal concordance in this AOP.\u003c/li\u003e\r\n\t\u003cli\u003eHowever, there is some uncertainty in the early manifestations of altered cardiovascular development and up-regulation of COX-2. It is possible that the first manifestations of altered cardiovascular development result from mechanisms other than COX-2. For example, sex determining region Y-box-9b (Sox9b) is first expressed in zebrafish at around 24 hpf and has been known to cause some altered cardiovascular phenotypes (Hofsteen et al 2013; Li et al 2002). However, no studies have yet investigated temporal concordance of regulation of Sox9b by the AhR prior to 72 hpf (Hofsteen et al 2013). It is also possible that temporal concordance of early increases in COX-2 is obscured by the relatively little fold-changes observed for COX-2. Additional investigations into up-regulation of COX-2 by activation of the AhR across developmental stages is warranted.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConsistency:\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThere are no known AhR-mediated effects that occur at concentrations below those that cause alteration in cardiovascular development and function that result in early life stage mortality in fishes, amphibians, reptiles, or birds.\u003c/li\u003e\r\n\t\u003cli\u003eThere are no studies in which COX-2 and altered cardiovascular development or function were co-investigated in which altered cardiovascular development or function occurred without an up-regulation in COX-2.\u003c/li\u003e\r\n\t\u003cli\u003eIn TCDD exposure groups, some individuals do not manifest alterations in cardiovascular development or function (Dong et al 2010). TCDD exposed individuals of Japanese medaka that did not manifest alterations in cardiovascular development or function had expression of COX-2 that was not statistically different than controls, while individuals that did manifest alterations in cardiovascular development or function had increased expression of COX-2 (Dong et al 2010).\u003c/li\u003e\r\n\t\u003cli\u003eThere is also consistency in the TCDD-induced alterations in cardiovascular phenotype between distantly related oviparous taxa, namely fish and birds (Teraoka et al 2008; Fujisawa et al 2014). Likewise, COX-2 is known to be up-regulated in both these taxa (Teraoka et al 2008; Fujisawa et al 2014). Cardiovascular development and function and COX-2 have not been investigated in amphibians or reptiles.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eUncertainties, inconsistencies, and data gaps:\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThere are several other pathways by which activation of the AhR could result in altered cardiovascular development and function in developing embryos. These include, but are not limited to, down-regulation in Sox9b, BMP-4, and genes in the cell cycle gene cluster (Hofsteen et al 2013; Jonsson et al 2007), oxidative stress (Goldstone \u0026amp; Stegeman 2008), and AhR cross-talk with hypoxia inducible factor 1\u0026alpha; (HIF1\u0026alpha;) causing reduced transcription of vascular endothelial growth factor (VEGF) (\u003ca href=\"https://aopwiki.org/aops/150\"\u003eAOP 150\u003c/a\u003e).\u003c/li\u003e\r\n\t\u003cli\u003eInvestigations of knockdown and null strains for Sox9b in zebrafish do not result in the complete phenotype of altered cardiovascular development recorded in embryos following exposure to planar aromatic hydrocarbons (Hofsteen et al 2013). Specifically, knockdown or knockout of Sox9b is associated with mild pericardial edema, unlooping, loss of proepicardium, and failure to form epicardium and endocardial cushions, but does not result in typical TCDD-mediated effects of a compacted ventricle or an\u0026nbsp;elongated string-like atrium (Hofsteen et al 2013). Altered cardiovascular development as a result of complete knockout of\u0026nbsp;Sox9b is not severe enough to cause complete cardiac failure and early life stage mortality in zebrafish (Hofsteen et al 2013). Injection of TCDD exposed embryos with Sox9b mRNA was able to prevent the Sox9b phenotype of cardiovascular development, however it did not prevent altered cardiovascular development altogether (Hofsteen et al 2013).Considering,\u0026nbsp;TCDD is only able to decrease expression of Sox9b in the heart by up to about 50% and complete knockout of\u0026nbsp;Sox9b expression is not lethal suggests that Sox9b is not essential to TCDD-mediated alteration in cardiovascular development and function\u0026nbsp;(Hofsteen et al 2013).\u003c/li\u003e\r\n\t\u003cli\u003eOxidative stress as a result of induction in CYP1A has commonly been proposed as the mechanism of altered cardiovascular development and function and CYP1A follows dose- and temporal concordance with mortality across numerous investigations in fishes and birds (Goldstone \u0026amp; Stegeman 2008). Early studies of CYP1A knockdown in zebrafish demonstrated protection against alteration in cardiovascular development and function induced by exposure to TCDD (Teraoka et al 2003). However, more recent investigations have observed no protection (Carney et al 2006). This inconsistency has been proposed to result from the earlier studies only recording alteration in cardiovascular development and function at early stages when adverse effects are difficult to accurately observe (Carney et al 2006). In birds, COX-2 inhibitors have no effect on expression of CYP1A but protect against TCDD induced alteration in cardiovascular development and function suggesting that CYP1A is not involved in toxicities (Fujisawa et al 2014).\u003c/li\u003e\r\n\t\u003cli\u003eFor cross-species and cross-taxa extrapolation, there is uncertainty in whether COX-2 is up-regulated by AhR through genomic or non-genomic mechanisms. Specifically, there is no detailed analysis regarding how widespread COX-2 genes which contain DREs in the promoter region are among species and among taxa and whether non-genomic or genomic mechanisms of up-regulation in COX-2 are more ubiquitous.\u003c/li\u003e\r\n\t\u003cli\u003eAll mechanistic investigations into mechanisms of AhR-mediated alteration in cardiovascular development and function have been conducted in zebrafish, Japanese medaka, and chicken. Therefore, no mechanistic information is available to conclude cross-species extrapolation outside of a shared phenotype of altered cardiovascular development and function. There is no information about AhR-mediated alteration in cardiovascular development or function or up-regulation of COX-2 in early fishes (Petromyzontiformes; Myxiniformes; Chondrichthyes), amphibians, or reptiles.\u003c/li\u003e\r\n\t\u003cli\u003eDespite these uncertainties, the strong, quantitative link between activation of the AhR and early life stage mortality means that elucidating the\u0026nbsp;precise series of key events is less critical. Therefore, the evidence suggesting COX-2 as a primary mechanism\u0026nbsp;might be all that is necessary, although multiple mechanisms acting together is the most likely true mechanism.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","quantitative_considerations":"\u003cul\u003e\r\n\t\u003cli\u003eThe majority of the quantitative understanding and the strongest quantitative understanding is for the indirect relationship between activation of the AhR and early life stage mortality.\u003c/li\u003e\r\n\t\u003cli\u003eThere is a strong quantitative understanding between quantitative structure-activity relationship (QSAR) or binding affinity for the AhR and potency among PCDDs, PCDFs, and planar PCBs (Van den Berg et al 1998; 2006). Specifically, these studies have demonstrated that congeners with greater binding affinity have greater potency (Van den Berg et al 1998; 2006). This has partially contributed to the successful development of the toxic equivalency factor (TEF) methodology in risk assessment (Van den Berg et al 1998; 2006).\u003c/li\u003e\r\n\t\u003cli\u003eThere is also a strong quantitative understanding of differences in binding affinity of the AhR among species of birds and differences in sensitivity to early life stage mortality (Karchner et al 2006; Farmahin et al 2012; 2013; Manning et al 2013). Specifically, these studies demonstrate that species of birds with AhRs with greater affinity for DLCs have greater sensitivity than species with AhRs with lesser affinity for DLCs (Karchner et al 2006). These differences in sensitivity range by more than 40-fold for TCDD (Cohen-Barnhouse et al 2011). However, a quantitative understanding between differences in binding affinity of the AhR among species and differences in sensitivity to early life stage mortality is not yet available for other taxa (Doering et al 2013).\u003c/li\u003e\r\n\t\u003cli\u003eThere is some quantitative understanding between up-regulation of COX-2 and incidence of and severity of cardiac deformities for medakafish exposed to TCDD (Dong et al 2010). This quantitative understanding includes a strong linear relationship (R\u003csup\u003e2\u003c/sup\u003e = 0.88) between abundance of COX-2 transcript and heart area (Dong et al 2010). However, this information is not available with regards to multiple investigations, species, taxonomic groups, or chemicals.\u003c/li\u003e\r\n\t\u003cli\u003eThere is strong quantitative understanding between incidence of and severity of cardiovascular deformities and mortality. However, numerous different cardiovascular endpoints are investigated among studies making side-by-side comparisons difficult.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\u003c/ul\u003e\r\n","optional_considerations":"\u003cp\u003eThere are great differences in sensitivity to agonists of the AhR among species and among taxa and \u0026nbsp;great differences in potency among agonists of the AhR. Therefore, this AOP has utility towards the mechanistic understanding of adverse effects of agonists of the AhR with regard to cross-chemical, cross-species, and cross-taxa extrapolations. This utility has led to the development of a qAOP that has demonstrated utility in guiding more objective ecological risk assessments of native species to agonists of the AhR, particularly assessments of threatened or endangered species that often cannot be investigated in laboratory toxicity testing (Doering et al 2018).\u003c/p\u003e\r\n","references":"\u003cp\u003eAbnet, C.C.; Tanguay, R.L.; Heideman, W.; Peterson, R.E. 1999. Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: Greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners. Toxicol. Appl. Pharmacol\u003cem\u003e.\u003c/em\u003e 159, 41-51.\u003c/p\u003e\r\n\r\n\u003cp\u003eAntkiewicz, D.S.; Burns, C.G.; Carney, S.A.; Peterson, R.E.; Heideman, W. 2005. Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. 84, 368-377.\u003c/p\u003e\r\n\r\n\u003cp\u003eBak, S.M.; Lida, M.; Hirano, M.; Iwata, H.; Kim, E.Y. 2013. Potencies of red seabream AHR1- and AHR2-mediated transactivation by dioxins: implications of both AHRs in dioxin toxicity. Environ. Sci. Technol. 47 (6), 2877-2885.\u003c/p\u003e\r\n\r\n\u003cp\u003eBelair, C.D.; Peterson, R.E.; Heideman, W. (2001). Disruption of erythropoiesis by dioxin in the zebrafish. Dev. Dyn. 222 (4), 581-594.\u003c/p\u003e\r\n\r\n\u003cp\u003eBilliard, S.M.; Hahn, M.E.; Franks, D.G.; Peterson, R.E.; Bols, N.C.; Hodson, P.V. (2002). Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs). Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 133 (1), 55-68.\u003c/p\u003e\r\n\r\n\u003cp\u003eBrunstrom, B. (1990). Mono-ortho-chlorinated chlorobiphenyls: toxicity and induction of 7-ethoxyresorufin O-deethylase (EROD) activity in chick embryos. Arch. Toxicol. 64, 188-192.\u003c/p\u003e\r\n\r\n\u003cp\u003eBrunstrom, B.; Andersson, L. (1988). Toxicity and 7-ethoxyresorufin O-deethylase-inducing potency of coplanar polychlorinated biphenyls (PCBs) in chick embryos. Arch. Toxicol. 62, 263-266.\u003c/p\u003e\r\n\r\n\u003cp\u003eBuckler J.; Candrl, J.S.; McKee, M.J.; Papoulias, D.M.; Tillitt, D.E.; Galat, D.L. Sensitivity of shovelnose sturgeon (\u003cem\u003eScaphirhynchus platorynchus\u003c/em\u003e) and pallid sturgeon (\u003cem\u003eS. albus\u003c/em\u003e) early life stages to PCB-126 and 2,3,7,8-TCDD exposure. \u003cem\u003eEnviro. Toxicol. Chem. \u003c/em\u003e\u003cstrong\u003e2015\u003c/strong\u003e, 34(6), 1417-1424.\u003c/p\u003e\r\n\r\n\u003cp\u003eCanga, L., Paroli, L., Blanck, T. J., Silver, R. B., and Rifkind, A. B. (1993). 2,3,7,8-Tetrachlorodibenzo-p-dioxin increases cardiac myocyte intracellular calcium and progressively impairs ventricular contractile responses to isoproterenol and to calcium in chick embryo hearts. Mol. Pharmacol.\u0026nbsp; 44, 1142\u0026ndash;1151.\u003c/p\u003e\r\n\r\n\u003cp\u003eCarney, S.A.; Peterson, R.E.; Heideman, W. 2004. 2,3,7,8-tetrachlorodibenzo-p-dioxin activation of aryl hydrocarbon receptors/aryl hydrocarbon receptor nuclear translocator pathway causes developmental toxicity through a CYP1A-independent mechanism in zebrafish. Mol. Pharmacol. 66 (2), 512-521.\u003c/p\u003e\r\n\r\n\u003cp\u003eCarney, S.A.; Prasch, A.L.; Heideman, W.; Peterson, R.E. 2006. Understanding dioxin developmental toxicity using the zebrafish model. Birth Defects Research. A. 76, 7-18.\u003c/p\u003e\r\n\r\n\u003cp\u003eChen, G.; Bunce, N.J. (2003). Polybrominated diphenyl ethers as Ah receptor agonists and antagonists. Toxicol. Sci. 76 (2), 310-320.\u003c/p\u003e\r\n\r\n\u003cp\u003eClark, B.W.; Matson, C.W.; Jung, D.; Di Giulio, R.T. 2010. AHR2 mediates cardiac teratogenesis of polycyclic aromatic hydrocarbons and PCB-126 in Atlantic killifish (\u003cem\u003eFundulus heteroclitus\u003c/em\u003e). Aquat. Toxicol. 99, 232-240.\u003c/p\u003e\r\n\r\n\u003cp\u003eCohen-Barnhouse, A.M.; Zwiernik, M.J.; Link, J.E.; Fitzgerald, S.D.; Kennedy, S.W.; Herve, J.C.; Giesy, J.P.; Wiseman, S.; Yang, Y.; Jones, P.D.; Yi, W.; Collins, B.; Newsted, J.L.; Kay, D.; Bursian, S.J. 2011. Sensitivity of Japanese quail (\u003cem\u003eCoturnix japonica\u003c/em\u003e), common pheasant (\u003cem\u003ePhasianus colchicus\u003c/em\u003e), and white leghorn chicken (\u003cem\u003eGallus gallus domesticus\u003c/em\u003e) embryos to \u003cem\u003ein ovo\u003c/em\u003e exposure to TCDD, PeCDF, and TCDF. Toxicol. Sci. 119, 93-102.\u003c/p\u003e\r\n\r\n\u003cp\u003eCook, P.M.; Robbins, J.A.; Endicott, D.D.; Lodge, K.B.; Guiney, P.D.; Walker, M.K.; Zabel, E.W.; Peterson, R.E. 2003. Effects of aryl hydrocarbon receptor-mediated early life stage toxicity on lake trout populations in Lake Ontario during the 20\u003csup\u003eth\u003c/sup\u003e century. Enviro. Sci. Technol. 37 (17), 3864-3877.\u003c/p\u003e\r\n\r\n\u003cp\u003eDegner, S.C.; Kemp, M.Q.; Hockings, J.K.; Romagnolo, D.F. (2007). Cyclooxygenase-2 promoter activation by the aromatic hydrocarbon receptor in breast cancer MCF-7 cells: Repressive effects of conjugated linoleic acid. Nutri. Canc. 56 (2), 248-257.\u003c/p\u003e\r\n\r\n\u003cp\u003eDenison, M.S.; Heath-Pagliuso, S. The Ah receptor: a regulator of the biochemical and toxicological actions of structurally diverse chemicals. Bull. Environ. Contam. Toxicol. \u003cstrong\u003e1998\u003c/strong\u003e, 61 (5), 557-568.\u003c/p\u003e\r\n\r\n\u003cp\u003eDoering, J.A.; Giesy, J.P.; Wiseman, S.; Hecker, M. Predicting the sensitivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain. \u003cem\u003eEnviron. Sci. Pollut. Res. Int.\u003c/em\u003e \u003cstrong\u003e2013\u003c/strong\u003e, 20(3), 1219-1224.\u003c/p\u003e\r\n\r\n\u003cp\u003eDoering, J.A.; Wiseman, S; Beitel, S.C.; Giesy, J.P.; Hecker, M. 2014. Identification and expression of aryl hydrocarbon receptors (AhR1 and AhR2) provide insight in an evolutionary context regarding sensitivity of white sturgeon (\u003cem\u003eAcipenser transmontanus\u003c/em\u003e) to dioxin-like compounds. Aquat. Toxicol. 150, 27-35.\u003c/p\u003e\r\n\r\n\u003cp\u003eDoering, J.A.; Wiseman, S.; Giesy, J.P.; Hecjer, M. 2018. A cross-species quantitative adverse outcome pathway for activation of the aryl hydrocarbon receptor leading to early life stage mortality in birds and fishes. Environ. Sci. Technol. 52 (13), 7524-7533.\u003c/p\u003e\r\n\r\n\u003cp\u003eDong, W.; Matsumura, F.; Kullman, S.W. (2010). TCDD induced pericardial edema and relative COX-2 expression in medaka (Oryzias latipes) embryos. Toxicol. Sci. 118 (1), 213-223.\u003c/p\u003e\r\n\r\n\u003cp\u003eDuncan, D.M.; Burgess, E.A.; Duncan, I. 1998. Control of distal antennal identity and tarsal development in Drosophila by spineless-aristapedia, a homolog of the mammalian dioxin receptor. Genes Dev. 12, 1290-1303.\u003c/p\u003e\r\n\r\n\u003cp\u003eElonen, G.E.; Spehar, R.L.; Holcombe, G.W.; Johnson, R.D.; Fernandez, J.D.; Erickson, R.J.; Tietge, J.E.; Cook, P.M. Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-\u003cem\u003ep\u003c/em\u003e-dioxin to seven freshwater fish species during early life-stage development.\u003cem\u003e Enviro. Toxico. Chem. \u003c/em\u003e\u003cstrong\u003e1998\u003c/strong\u003e, 17, 472-483.\u003c/p\u003e\r\n\r\n\u003cp\u003eEmmons, R.B.; Duncan, D.; Estes, P.A.; Kiefel, P.; Mosher, J.T.; Sonnenfeld, M.; Ward, M.P.; Duncan, I.; Crews, S.T. 1999. The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila. Development. 126, 3937-3945.\u003c/p\u003e\r\n\r\n\u003cp\u003eFarmahin, R.; Crump, D.; O\u0026rsquo;Brien, J.M.; Jones, S.P.; Kennedy, S.W. (2016). Time-dependent transcriptomic and biochemical responses of 6-formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are explained by AHR activation time. Biochem. Pharmacol. 115 (1), 134-143.\u003c/p\u003e\r\n\r\n\u003cp\u003eFarmahin, R.; Manning, G.E.; Crump, D.; Wu, D.; Mundy, L.J.; Jones, S.P.; Hahn, M.E.; Karchner, S.I.; Giesy, J.P.; Bursian, S.J.; Zwiernik, M.J.; Fredricks, T.B.; Kennedy, S.W. 2013. Amino acid sequence of the ligand-binding domain of the aryl hydrocarbon receptor 1 predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol. Sci. 131 (1), 139-152.\u003c/p\u003e\r\n\r\n\u003cp\u003eFarmahin, R.; Wu, D.; Crump, D.; Herve, J.C.; Jones, S.P.; Hahn, M.E.; Karchner, S.I.; Giesy, J.P.; Bursian, S.J.; Zwiernik, M.J.; Kennedy, S.W. 2012. Sequence and \u003cem\u003ein vitro\u003c/em\u003e function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict \u003cem\u003ein vivo\u003c/em\u003e sensitivity to dioxins. Enviro. Sci. Toxicol. 46 (5), 2967-2975.\u003c/p\u003e\r\n\r\n\u003cp\u003eFujisaw, N.; Nakayama, S.M.M.; Ikenaka, Y.; Ishizuka, M. 2014. TCDD-induced chick cardiotoxicity is abolished by a selective cyclooxygenase-2 (COX-2) inhibitor NS398. Arch. Toxicol. 88, 1739-1748.\u003c/p\u003e\r\n\r\n\u003cp\u003eGiesy, J.P.; Jones, P.D.; Kannan, K.; Newstead, J.L.; Tillitt, D.E.; Williams, L.L. Effects of chronic dietary exposure to environmentally relevant concentrations to 2,3,7,8-tetrachlorodibenzo-\u003cem\u003ep\u003c/em\u003e-dioxin on survival, growth, reproduction and biochemical responses of female rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e). Aquat. Toxicol. \u003cstrong\u003e2002\u003c/strong\u003e, 59 (1-2), 35-53.\u003c/p\u003e\r\n\r\n\u003cp\u003eGoldstone, H.M.; Stegeman, J.J. 2008. Molecular mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin cardiovascular embryotoxicity. Drug. Metab. Rev. 38 (1), 261-289.\u003c/p\u003e\r\n\r\n\u003cp\u003eHahn, M.E. 2002. Aryl hydrocarbon receptors: diversity and evolution. Chemico-Biol. Interact. 141, 131-160.\u003c/p\u003e\r\n\r\n\u003cp\u003eHahn, M.E.; Karchner, S.I.; Evans, B.R.; Franks, D.G.; Merson, R.R.; Lapseritis, J.M. 2006. Unexpected diversity of aryl hydrocarbon receptors in non-mammalian vertebrates: Insights from comparative genomics. J. Exp. Zool. A. Comp. Exp. Biol. 305, 693-706.\u003c/p\u003e\r\n\r\n\u003cp\u003eHahn, M.E.; Poland, A.; Glover, E.; Stegeman, J.J. 1994. Photoaffinity labeling of the Ah receptor: phylogenetic survey of diverse vertebrate and invertebrate species. Arch. Biochem. Biophys. 310, 218-228.\u003c/p\u003e\r\n\r\n\u003cp\u003eHahn, M.E.; Woodlin, B.R.; Stegeman, J.J.; Tillitt, D.E. 1998. Aryl hydrocarbon receptor function in early vertebrates: Inducibility of cytochrome P450 1A in agnathan and elasmobranch fish. Comp. Biochem. Physiol. C. 120, 67-75.\u003c/p\u003e\r\n\r\n\u003cp\u003eHansson, M.C.; Hahn, M.E. 2008. Functional properties of the four Atlantic salmon (\u003cem\u003eSalmo salar\u003c/em\u003e) aryl hydrocarbon receptor type 2 (AHR2) isoforms. Aquat. Toxicol. 86, 121-130.\u003c/p\u003e\r\n\r\n\u003cp\u003eHansson, M.C.; Wittzell, H.; Persson, K.; von Schantz, T. 2004. Unprecedented genomic diversity of AhR1 and AhR2 genes in Atlantic salmon (\u003cem\u003eSalmo salar \u003c/em\u003eL.). Aquat. Toxicol. 68 (3), 219-232.\u003c/p\u003e\r\n\r\n\u003cp\u003eHeid, S. E., Walker, M. K., and Swanson, H. I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci.\u0026nbsp; 61, 187\u0026ndash;196.\u003c/p\u003e\r\n\r\n\u003cp\u003eHengstler, J.G.; Van der Burg, B.; Steinberg, P.; Oesch, F. Interspecies differences in cancer susceptibility and toxicity. \u003cem\u003eDrug. Metab. Rev. \u003c/em\u003e\u003cstrong\u003e1999\u003c/strong\u003e, 31, 917-970.\u003c/p\u003e\r\n\r\n\u003cp\u003eHill, A.J.; Bello, S.M.; Prasch, A.L.; Peterson, R.E.; Heideman, W. (2004). Water permeability and TCDD-induced edema in zebrafish early-life stages. Toxicol. Sci. 100, 486-494.\u003c/p\u003e\r\n\r\n\u003cp\u003eHoffman, D.J., Rice, C.P., Kubiak, T.J., 1996. PCBs and dioxins in birds. In: Beyer, W.N.,\u003c/p\u003e\r\n\r\n\u003cp\u003eHeinz, G.H., Redmon-Norwood, A.W. (Eds.), Environmental Contaminants in Wildlife:\u003c/p\u003e\r\n\r\n\u003cp\u003eInterpreting Tissue Concentrations. CRC Press, pp. 165\u0026ndash;207.\u003c/p\u003e\r\n\r\n\u003cp\u003eHoffman, D.J., Melancon, M.J., Klein, P.N., Eisemann, J.D., Spann, J.W., 1998. Comparative\u003c/p\u003e\r\n\r\n\u003cp\u003edevelopmental toxicity of planar polychlorinated biphenyl congeners in chickens,\u003c/p\u003e\r\n\r\n\u003cp\u003eAmerican kestrels, and common terns. Environ. Toxicol. Chem. 17, 747\u0026ndash;757.\u003c/p\u003e\r\n\r\n\u003cp\u003eHofsteen, P.; Plavicki, J.; Johnson, S.D.; Peterson, R.E.; Heideman, W. Sox9b is required for epicardium formation and plays a role in TCDD-induced heart malformation in zebrafish. Molec. Pharmacol. 2013, 84, 353-360.\u003c/p\u003e\r\n\r\n\u003cp\u003eHuang, C.C.; Chen, P.C.; Huang, C.W.; Yu, J. (2007). Aristolochic acid induces heart failure in zebrafish embryos that is mediated by inflammation. Toxicol. Sci. 100, 486-494.\u003c/p\u003e\r\n\r\n\u003cp\u003eHuang, L.; Wang, C.; Zhang, Y.; Li, J.; Zhong, Y.; Zhou, Y.; Chen, Y.; Zuo, Z. (2012). Benzo[a]pyrene exposure influences the cardiac development and the expression of cardiovascular relative genes in zebrafish (Daniorerio) embryos. Chemosphere. 87 (4), 369-375.\u003c/p\u003e\r\n\r\n\u003cp\u003eIvnitski, I., Elmaoued, R., and Walker, M. K. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary development is preceded by a decrease in myocyte proliferation and an increase in cardiac apoptosis. Teratology\u0026nbsp; 64, 201\u0026ndash;212.\u003c/p\u003e\r\n\r\n\u003cp\u003eJohnson, R.D.; Tietge, J.E.; Jensen, K.M.; Fernandez, J.D.; Linnum, A.L.; Lothenbach, D.B.; Holcombe, G.W.; Cook, P.M.; Christ, S.A.; Lattier, D.L.; Gordon, D.A. Toxicity of 2,3,7,8-tetrachlorodibenzo-\u003cem\u003ep\u003c/em\u003e-dioxin to early life stage brooke trout (\u003cem\u003eSalvelinus fontinalis\u003c/em\u003e) following parental dietary exposure. \u003cem\u003eEnviro. Toxicol. Chem.\u003c/em\u003e \u003cstrong\u003e1998\u003c/strong\u003e, 17 (12), 2408-2421.\u003c/p\u003e\r\n\r\n\u003cp\u003eJonsson, M.E.; Jenny, M.J.; Woodin, B.R.; Hahn, M.E.; Stegeman, J.J. (2007). Role of AHR2 in the expression of novel cytochrome P450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3\u0026rsquo;,4,4\u0026rsquo;,5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Sci. 100 (1), 180-193.\u003c/p\u003e\r\n\r\n\u003cp\u003eJonsson, M.E.; Kubota, A.; Timme-Laragy, A.R.; Woodin, B.; Stegeman, J.J. (2012). Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish. Toxicol. Appl. Pharmacol. 265 (2), 166-174.\u003c/p\u003e\r\n\r\n\u003cp\u003eJung, R.E.; Walker, M.K. (1997). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on development of anuran amphibians. Enviro. Toxicol. Chem. 16 (2), 230-240.\u003c/p\u003e\r\n\r\n\u003cp\u003eKarchner, S.I.; Franks, D.G.; Kennedy, S.W.; Hahn, M.E. 2006. The molecular basis for differential dioxin sensitivity in birds: Role of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. USA. 103, 6252-6257.\u003c/p\u003e\r\n\r\n\u003cp\u003eKarchner, S.I.; Powell, W.H.; Hahn, M.E. 1999. Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the Teleost \u003cem\u003eFundulus heteroclitus\u003c/em\u003e. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors. J. Biol. Chem. 274, 33814-33824.\u003c/p\u003e\r\n\r\n\u003cp\u003eKardong, K.V. (2006). Vertebrates: comparative anatomy, function, evolution. McGraw-Hill Higher Eduction. Boston, USA.\u003c/p\u003e\r\n\r\n\u003cp\u003eKleeman, J.M.; Olson, J.R.; Peterson, R.E. Species differences in 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity and biotransformation in fish. \u003cem\u003eFundam. Appl. Toxicol. \u003c/em\u003e\u003cstrong\u003e1988\u003c/strong\u003e, 10(2), 206-213.\u003c/p\u003e\r\n\r\n\u003cp\u003eKopf, P.G.; Walker, M.K. (2009). Overview of developmental heart defects by dioxins, PCBs, and pesticides. J. Environ. Sci. Health C. Environ. Carcinog. Ecotoxicol. Rev. 27 94), 276-285.\u003c/p\u003e\r\n\r\n\u003cp\u003eKorkalainen, M.; Tuomisto, J.; Pohjanvirta, R. The AH receptor of the most dioxin-sensitive specie, guinea pig, is highly homologous to the human AH receptor. \u003cem\u003eBiochem. Biophys. Res. Commun. \u003c/em\u003e\u003cstrong\u003e2001\u003c/strong\u003e, 285, 1121-1129.\u003c/p\u003e\r\n\r\n\u003cp\u003eLahvis, G.P.; Bradfield, C.A. 1998. Ahr null alleles: distinctive or different? Biochem. Pharmacol. 56, 781-787.\u003c/p\u003e\r\n\r\n\u003cp\u003eLavine, J.A.; Rowatt, A.J.; Klimova, T.; Whitington, A.J.; Dengler, E.; Beck, C.; Powell, W.H. 2005. Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Sci. 88 (1), 60-72.\u003c/p\u003e\r\n\r\n\u003cp\u003eLanham, K.A.; Peterson, R.E.; Heideman, W. (2012). Sensitivity to dioxin decreases as zebrafish mature. Toxicol. Sci. 127 (2), 360-370.\u003c/p\u003e\r\n\r\n\u003cp\u003eLi, M.; Zhao, C.; Wang, Y.; Zhao, Z.; Meng, A. (2002). Zebrafish sox9b is an early neural crest marker. Dev. Genes Evol. 212, 203-206.\u003c/p\u003e\r\n\r\n\u003cp\u003eManning G.E.; Farmahin, R.; Crump, D.; Jones, S.P.; Klein, J.; Konstantinov, A.; Potter, D.; Kennedy, S.W. 2012. A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD50 of polychlorinated biphenyls in avian species. Toxicol. Appl. Pharm. 263, 390-401.\u003c/p\u003e\r\n\r\n\u003cp\u003eMurk, A.J.; Legler, J.; Denison, M.S.; Giesy, J.P.; Van De Guchte, C.; Brouwer, A. (1996). Chemical-activated luciferase gene expression (CALUX): A novel in vitro bioassay for Ah receptor active compounds in sediments and pore water. Toxicol. Sci. 33 (1), 149-160.\u003c/p\u003e\r\n\r\n\u003cp\u003eNacci, D.E. Champlin, D.; Jayaraman, S. (2010). Adaptation of the estuarine fish Fundulus heteroclitus (Atlantic killifish) to polychlorinated biphenyls (PCBs). Estuaries and Coasts. 33 (4), 853-864.\u003c/p\u003e\r\n\r\n\u003cp\u003eOhi, H.; Fujita, Y.; Miyao, M.; Saguchi, K.; Murayama, N.; Higuchi, S. 2003. Molecular cloning and expression analysis of the aryl hydrocarbon receptor of Xenopus laevis. Biochem. Biophysic. Res. Comm. 307 (3), 595-599.\u003c/p\u003e\r\n\r\n\u003cp\u003eOkey, A.B. An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XI. \u003cem\u003eToxicol. Sci.\u003c/em\u003e \u003cstrong\u003e2007\u003c/strong\u003e, 98, 5-38.\u003c/p\u003e\r\n\r\n\u003cp\u003ePark, Y.J.; Lee, M.J.; Kim, H.R.; Chung, K.H.; Oh, S.M. Developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in artificially fertilized crucian carp (Carassius auratus) embryo. \u003cem\u003eSci. Totl. Enviro.\u003c/em\u003e \u003cstrong\u003e2014\u003c/strong\u003e, 491-492, 271-278.\u003c/p\u003e\r\n\r\n\u003cp\u003ePongratz, I.; Mason, G.G.; Poellinger, L. Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. J. Biol. Chem. 1992, 267 (19), 13728-13734.\u003c/p\u003e\r\n\r\n\u003cp\u003ePowell, D.C., Aulerich, R.J., Meadows, J.C., Tillitt, D.E., Kelly, M.E., Stromborg, K.L.,\u003c/p\u003e\r\n\r\n\u003cp\u003eMelancon, M.J., Fitzgerald, S.D., Bursian, S.J., 1998. Effects of 3,3\u0026prime;,4,4\u0026prime;,5-\u003c/p\u003e\r\n\r\n\u003cp\u003epentachlorobiphenyl and 2,3,7,8-tetrachlorodibenzo-p-dioxin injected into the\u003c/p\u003e\r\n\r\n\u003cp\u003eyolks of double-crested cormorant (Phalacrocorax auritus) eggs prior to incubation.\u003c/p\u003e\r\n\r\n\u003cp\u003eEnviron. Toxicol. Chem. 17, 2035\u0026ndash;2040.\u003c/p\u003e\r\n\r\n\u003cp\u003ePrasch, A.L.; Teraoka, H.; Carney, S.A.; Dong, W.; Hiraga, T.; Stegeman, J.J.; Heideman, W.; Peterson, R.E. 2003. Toxicol. Sci. Aryl hydrocarbon receptor 2 mediated 2,3,7,8-tetrachlorodibenzo-\u003cem\u003ep\u003c/em\u003e-dioxin developmental toxicity in zebrafish. 76 (1), 138-150.\u003c/p\u003e\r\n\r\n\u003cp\u003eScott, J.A.; Incardona, J.P.; Pelkki, K.; Shepardson, S.; Hodson, P.V. (2011). AhR2-mediated, CYP1A-independent cardiovascular toxicity in zebrafish (Danio rerio) embryos exposed to retene. Aquat. Toxicol. 101 (1), 165-174.\u003c/p\u003e\r\n\r\n\u003cp\u003eShoots, J.; Fraccalvieri, D.; Franks, D.G.; Denison, M.S.; Hahn, M.E.; Bonati, L.; Powell, W.H. 2015. An aryl hydrocarbon receptor from the salamander Ambystoma mexicanum exhibits low sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enviro. Sci. Technol\u003cem\u003e. \u003c/em\u003e49, 6993-7001.\u003c/p\u003e\r\n\r\n\u003cp\u003eSpitsbergen, J.M.; Kleeman, J.M.; Peterson, R.E. 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in yellow perch (\u003cem\u003ePerca flavescens\u003c/em\u003e). \u003cem\u003eJ. Toxicol. Environ. Health\u003c/em\u003e. \u003cstrong\u003e1988\u003c/strong\u003e, 23, 359-383.\u003c/p\u003e\r\n\r\n\u003cp\u003eSpitsbergen, J.M.; Kleeman, J.M.; Peterson, R.E. Morphologic lesions and acute toxicity in rainbow trout (\u003cem\u003eSalmo gairdneri\u003c/em\u003e) treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. \u003cem\u003eJ. Toxicol. Environ. Health. \u003c/em\u003e\u003cstrong\u003e1988\u003c/strong\u003e, 23, 333-358.\u003c/p\u003e\r\n\r\n\u003cp\u003eSpitsbergen, J.M.; Schat, K.A.; Kleeman, J.M.; Peterson, R.E. Interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with immune responses of rainbow trout. \u003cem\u003eVet. Immunol. Immunopathol. \u003c/em\u003e\u003cstrong\u003e1986\u003c/strong\u003e, 12(1-4), 263-280.\u003c/p\u003e\r\n\r\n\u003cp\u003eTanguay, R.L.; Abnet, C.C.; Heideman, W. Peterson, R.E. (1999). Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor1. Biochimica et Biophysica Act 1444, 35-48.\u003c/p\u003e\r\n\r\n\u003cp\u003eTeraoka, H.; Dong, W.; Tsujimoto, Y.; Iwasa, H.; Endoh, D.; Ueno, N.; Stegeman, J.J.; Peterson, R.E.; Hiraga, T. (2003). Induction of cytochrome P450 1A is required for circulatory failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish. Biochem. Biophys. Res. Commun. 304, 223-228.\u003c/p\u003e\r\n\r\n\u003cp\u003eTeraoka, H.; Kubota, A.; Kawai, Y.; Hiraga, T. (2008). Prostanoid signaling mediates circulation failure caused by TCDD in developing zebrafish. Interdis. Studies Environ. Chem. Biol. Resp. Chem. Pollut. 61-80.\u003c/p\u003e\r\n\r\n\u003cp\u003eTeraoka, H.; Okuno, Y.; Nijoukubo, D.; Yamakoshi, A.; Peterson, R.E.; Stegeman, J.J.; Kitazawa, T.; Hiraga, T.; Kubota, A. (2014). Involvement of COX2-thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish. Aquat. Toxicol. 154, 19-25.\u003c/p\u003e\r\n\r\n\u003cp\u003eThackaberry, E.A.; Nunez, B.A.; Ivnitski-Steele, I.D. Friggins, M.; Walker, M.K. (2005). Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on murine heart development: Alteration in fetal and postnatal cardiac growth, and postnatal cardiac chronotropy. Toxicol. Sci. 88 (1), 242-249.\u003c/p\u003e\r\n\r\n\u003cp\u003eTillitt, D.E.; Buckler, J.A.; Nicks, D.K.; Candrl, J.S.; Claunch, R.A.; Gale, R.W.; Puglis, H.J.; Little, E.E.; Linbo, T.L.; Baker, M. Sensitivity of lake sturgeon (Acipenser fulvescens) early life stages to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,3\u0026rsquo;,4,4\u0026rsquo;,5-pentachlorobiphenyl. 2015. Enviro. Toxicol. Chem. DOI: 10.1002/etc.3614.\u003c/p\u003e\r\n\r\n\u003cp\u003eToomey, B.H.; Bello, S.; Hahn, M.E.; Cantrell, S.; Wright, P.; Tillitt, D.; Di Giulio, R.T. TCDD induces apoptotic cell death and cytochrome P4501A expression in developing \u003cem\u003eFundulus heteroclitus\u003c/em\u003e embryos. \u003cem\u003eAquat. Toxicol.\u003c/em\u003e \u003cstrong\u003e2001\u003c/strong\u003e, 53, 127-138.\u003c/p\u003e\r\n\r\n\u003cp\u003eUno, S.; Dalton, T.P.; Sinclair, P.R.; Gorman, N.; Wang, B.; Smith, A.G.; Miller, M.L.; Shertzer, H.G.; Nebert, D.W. (2004). Cyp1a1 (-/-) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria. Toxicol. Appl. Pharmacol. 196 (3), 410-421.\u003c/p\u003e\r\n\r\n\u003cp\u003eVan den Berg, M.; Birnbaum, L.; Bosveld, A.T.C.; Brunstrom, B.; Cook, P.; Feeley, M.; Giesy, J.P.; Hanberg, A.; Hasegawa, R.; Kennedy, S.W.; Kubiak, T.; Larsen, J.C.; van Leeuwen, R.X.R.; Liem, A.K.D.; Nolt, C.; Peterson, R.E.; Poellinger, L.; Safe, S.; Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PECDFs for human and wildlife. Enviro. Hlth. Persp. \u003cstrong\u003e1998\u003c/strong\u003e, 106, 775-792.\u003c/p\u003e\r\n\r\n\u003cp\u003eVan den Berg, M.; Birnbaum, L.S.; Dension, M.; De Vito, M.; Farland, W.; Feeley, M.; Fiedler, H.; Hakansson, H.; Hanberg, A.; Haws, L.; Rose, M.; Safe, S.; Schrenk, D.; Tohyama, C.; Tritscher, A.; Tuomisto, J.; Tysklind, M.; Walker, N.; Peterson, R.E. 2006. The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. 93 (2), 223-241.\u003c/p\u003e\r\n\r\n\u003cp\u003eVan Tiem, L.A.; Di Giulio, R.T. 2011. AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e). Toxicol. Appl. Pharmacol. 254 (3), 280-287.\u003c/p\u003e\r\n\r\n\u003cp\u003eWalker, M.K.; Catron, T.F. (2000). Characterization of cardiotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related chemicals during early chick embryo development. Toxicol. Appl. Pharmacol. 167 (3), 210-221.\u003c/p\u003e\r\n\r\n\u003cp\u003eWalker, M.K.; Spitsbergen, J.M.; Olson, J.R.; Peterson, R.E. 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) toxicity during early life stage development of lake trout (\u003cem\u003eSalvelinus namaycush\u003c/em\u003e). \u003cem\u003eCanad. J. Fisheries Aqua. Sci.\u003c/em\u003e \u003cstrong\u003e1991\u003c/strong\u003e, 48, 875-883.\u003c/p\u003e\r\n\r\n\u003cp\u003eWaller, C.L.; McKinney, J.D. (1992). Comparative molecular field analysis of polyhalogenated dibenzo-p-dioixns, dibenzofurans, and biphenyls. J. Med. Chem. 35, 3660-2666.\u003c/p\u003e\r\n\r\n\u003cp\u003eWaller, C.; McKinney, J. (1995). Three-dimensional quantitative structure-activity relationships of dioxins and dioxin-like compounds: model validation and Ah receptor characterization. Chem. Res. Toxicol. 8, 847-858.\u003c/p\u003e\r\n\r\n\u003cp\u003eWalter, G.L.; Jones, P.D.; Giesy, J.P. Pathologic alterations in adult rainbow trout, \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e, exposed to dietary 2,3,7,8-tetrachlorodibenzo-p-dioxin. \u003cem\u003eAquat. Toxicol.\u003c/em\u003e \u003cstrong\u003e2000\u003c/strong\u003e, 50, 287-299.\u003c/p\u003e\r\n\r\n\u003cp\u003eWhitlock, J.P.; Okino, S.T.; Dong, L.Q.; Ko, H.S.P.; Clarke Katzenberg, R.; Qiang, M.; Li, W. 1996. Induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. Faseb. J. 10, 809-818.\u003c/p\u003e\r\n\r\n\u003cp\u003eWhyte, J.J.; Jung, R.E.; Schmitt, C.J.; Tillitt, D.E. (2008). Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit. Rev. Toxicol. 30 (4), 347-570.\u003c/p\u003e\r\n\r\n\u003cp\u003eWirgin, I.; Roy, N.K.; Loftus, M.; Chambers, R.C.; Franks, D.G.; Hahn, M.E. 2011. Mechanistic basis of resistance to PCBs in Atlantic tomcod from the Hudson River. Science. 331, 1322-1324.\u003c/p\u003e\r\n\r\n\u003cp\u003eYamauchi, M.; Kim, E.Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2,3,7,8-tetrachlorodibenzo-\u003cem\u003ep\u003c/em\u003e-dioxin (TCDD) in developing red seabream (\u003cem\u003ePagrus major\u003c/em\u003e) embryos: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. \u003cem\u003eAquat. Toxicol.\u003c/em\u003e \u003cstrong\u003e2006\u003c/strong\u003e, 16, 166-179.\u003c/p\u003e\r\n\r\n\u003cp\u003eZabel, E.W; Cook, P.M.; Peterson, R.E. Toxic equivalency factors of polychlorinated dibenzo-p-dioxin, dibenzofuran and biphenyl congeners based on early-life stage mortality in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol. \u003cstrong\u003e1995\u003c/strong\u003e. 31, 315-328.\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003e\u003cstrong\u003eAssessment of WoE calls:\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eActivation, AhR leads to dimerization, AHR/ARNT\u003c/strong\u003e: High\u003c/p\u003e\r\n\r\n\u003cp\u003eRationale: The call of \u0026#39;High\u0026#39; is based on overwhelming empirical\u0026nbsp;evidence in numerous species of mammals, birds, amphibians, and fishes. Further, because of overwhelming evidence of essentiality based on targeted knockdown/knockout studies. No uncertainties or inconsistencies are known which affect the WoE call.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eDimerization, AHR/ARNT leads to increase, COX-2 expression\u003c/strong\u003e: High\u003c/p\u003e\r\n\r\n\u003cp\u003eRationale: The call of \u0026quot;High\u0026quot; is based on convincing empirical evidence in three species (two fish and one bird). Further, because of convincing biological plausibility based on identification of dioxin-response elements in the promoter region of COX-2. Uncertainties and inconsistencies are only related to lack of any information on species outside of the three model species that have been investigated.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eIncrease, COX-2 expression leads to altered, cardiovascular development/function\u003c/strong\u003e: Moderate\u003c/p\u003e\r\n\r\n\u003cp\u003eRationale: The call of \u0026quot;Moderate\u0026quot; is based on overwhelming empirical\u0026nbsp;evidence and evidence of essentiality\u0026nbsp;in three species (two fish and one bird) based on studies using targeted knockdown of genes and selective agonists/antagonists. However, a lack of information on the role of COX-2 in cardiovascular development/function makes biological plausibility questionable at this time. Further, there is some uncertainty associated with pleiotropic effects of AhR activation and the high probability of multiple mechanisms acting concurrently to cause altered cardiovascular development/function.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eAltered, cardiovascular development/function leads to increase, early life stage mortality\u003c/strong\u003e; High\u003c/p\u003e\r\n\r\n\u003cp\u003eRationale: The call of \u0026quot;High\u0026quot; is based on overwhelming empirical evidence and biological plausibility in numerous species of mammals, birds, and fish. There are no known uncertainties or inconsistencies at this time.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eActivation, AhR leads to increase, early life stage mortality\u003c/strong\u003e: High\u003c/p\u003e\r\n\r\n\u003cp\u003eRationale: The call of \u0026quot;High\u0026quot; is based on overwhelming empirical\u0026nbsp;evidence and evidence of essentiality in numerous species of mammals, birds, amphibians, and fishes using regression analysis and targeted knockdown/knockout of AhR. There are no known uncertainties of inconsistences at this time.\u003c/p\u003e\r\n","background":"\u003cul\u003e\r\n\t\u003cli\u003eThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family of proteins (Okey 2007). The AhR is a highly conserved and ancient protein with homologs having been identified in most major animal groups, apart from the most ancient lineages, such as sponges (Porifera) (Hahn et al 2002).\u003c/li\u003e\r\n\t\u003cli\u003eInvestigations of invertebrates possessing early homologs of the AhR suggest that the AhR evolutionarily functioned in regulation of the cell cycle, cellular proliferation and differentiation, and cell-to-cell communications (Hahn et al 2002). However, critical functions in angiogenesis, regulation of the immune system, neuronal processes, metabolism, development of the heart and other organ systems, and detoxification have emerged sometime in early vertebrate evolution (Duncan et al\u0026nbsp;1998; Lahvis and Bradfield\u0026nbsp;1998; Emmons et al\u0026nbsp;1999).\u003c/li\u003e\r\n\t\u003cli\u003eActivation of the AhR by anthropogenic pollutants that act as agonists can result in a range of adverse biological effects. These effects can include hepatotoxicity, histological lesions, hemopoiesis, suppression of immune responses and healing, impaired reproductive and endocrine processes, teratogenesis, carcinogenesis, wasting syndrome, and mortality (Kleeman et al 1988; Spitsbergen et al 1986; Walter et al 2000; Giesy et al 2002; Spitsbergen et al 1988a; 1988b).\u003c/li\u003e\r\n\t\u003cli\u003eDespite the AhR being a highly conserved protein, differences in relative sensitivity to adverse effects both among and within vertebrate taxa are greater than 1000-fold (Cohen-Barnhouse et al 2011; Doering et al 2013; Hengstler et al 1999; Korkalainen et al 2001).\u003c/li\u003e\r\n\t\u003cli\u003eDifferences in binding affinity and transactivation of the AhR have been implicated as a key mechanism contributing to differences in sensitivity to agonists of the AhR among species and taxa. However, the precise mechanisms are not fully understood for all taxa.\u003c/li\u003e\r\n\t\u003cli\u003eHigh-throughput, next-generation \u0026lsquo;OMICs\u0026rsquo; technologies have identified hundreds to thousands of different genes that are regulated, either directly or indirectly, by the AhR (Brinkmann et al 2016; Doering et al 2016; Huang et al 2014; Li et al 2013; Whitehead et al 2010). These genes include Phase I and Phase II biotransformation enzymes, such as cytochrome P450 1A (CYP1A). Expressions and activities of CYP1A are routinely used as biomarkers of exposure to anthropogenic pollutants that act as agonists of the AhR (Whyte et al 2008).\u003c/li\u003e\r\n\t\u003cli\u003eOne gene which is regulated by AhR is cyclooxygenase-2 (COX-2) which is known to have roles in development of the heart in vertebrates (Dong et al 2010; Teraoka et al 2008; 2014). AhR-mediated dysregulation of COX-2 is associated with altered cardiovascular development, decreased blood flow, and cardiac failure causing mortality in early life stages of fish and birds\u0026nbsp;(Dong et al 2010; Teraoka et al 2008; 2014).\u003c/li\u003e\r\n\t\u003cli\u003eExposure to mixtures of agonists of the AhR during the 1950\u0026rsquo;s, 1960\u0026rsquo;s, and 1970\u0026rsquo;s has been implicated in early life stage mortality of Lake Ontario lake trout (\u003cem\u003eSalvelinus namaycush\u003c/em\u003e) leading to population collapse (Cook et al 2003). However, populations of mummichog (\u003cem\u003eFundulus heteroclitus\u003c/em\u003e) and Atlantic tomcod (\u003cem\u003eMicrogadus tomcod\u003c/em\u003e) exposed to lethal concentrations of agonists of the AhR have evolved tolerance through several mechanisms which has protected against population collapse (Nacci et al 2010; Wirgin et al 2011).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","user_defined_mie":"18: Activation, AhR","user_defined_ao":"947: Increase, early life stage mortality","oecd_project":"1.27","oecd_status_id":1,"graphical_representation_image_uid":"2017/04/18/3gysyjyu9l_AhR_agonism_embry_toxicity_figure_002_.pptx","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2018-03-20T15:57:25.000-04:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":18,"handbook_id":1,"project_129":false},{"id":23,"title":"Androgen receptor agonism leading to reproductive dysfunction (in repeat-spawning fish)","short_name":"Androgen receptor agonism leading to reproductive dysfunction","corresponding_author_id":313,"abstract":"\u003cp\u003eThis adverse outcome pathway details the linkage between binding and activation of androgen receptor as a nuclear transcription factor in females and reproductive dysfunction as evidenced through reductions cumulative fecundity and spawning in repeat-spawning fish species. \u0026nbsp;Androgen receptor mediated activities are one of the major activities of concern to endocrine disruptor screening programs worldwide. \u0026nbsp;Cumulative fecundity is the most apical endpoint considered in the OECD 229 Fish Short Term Reproduction Assay. The OECD 229 assay serves as screening assay for endocrine disruption and associated reproductive impairment (OECD 2012). Cumulative fecundity is one of several variables known to be of demographic significance in forecasting fish population trends. Therefore, this AOP has utility in supporting the application of measures of androgen receptor binding and activation as a nuclear transcription factor as a means to identify chemicals with known potential to adversely affect fish populations. At present this AOP is largely supported by evidence conducted with small laboratory model fish species such as \u003cem\u003ePimephales promelas\u003c/em\u003e, \u003cem\u003eOryzias latipes\u003c/em\u003e, and \u003cem\u003eFundulus heteroclitus\u003c/em\u003e. While many aspects of the biology underlying this AOP are largely conserved across vertebrates, particularly oviparous vertebrates, the relevance of this AOP to vertebrate classes other than fish as well as to fish species employing different reproductive strategies has not been established\u0026nbsp;at this time. Thus, caution should be used in applying this AOP beyond a fairly narrow range of fish species with life cycles similar to that of the three species noted above.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":1,"authors":"\u003cp\u003eDan Villeneuve, US EPA Mid-Continent Ecology Division (villeneuve.dan@epa.gov)\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003e\u003cstrong\u003eDomain(s) of Applicability\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eChemical\u003c/strong\u003e: This AOP applies to non-aromatizable androgens. Compounds which can bind the AR in vitro, but are converted to high potency estrogens in vivo through aromatization do not produce the profile of effects described in the present AOP (e.g., methyltestosterone [Ankley et al. 2001; Pawlowski et al. 2004]; androstenedione [OECD 2007]).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e: The AOP applies to females only.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eLife stages\u003c/strong\u003e: The relevant life stages for this AOP are reproductively mature adults. This AOP does not apply to adult stages that lack a sexually mature ovary, for example as a result of seasonal or environmentally-induced gonadal senescence (i.e., through control of temperature, photo-period, etc. in a laboratory setting).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eTaxonomic\u003c/strong\u003e: At present, the assumed taxonomic applicability domain of this AOP is iteroparous teleost fish species.\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eHowever, to date the majority of toxicological data on which this AOP is based has been limited to several small fish species, fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), and mummichog (Fundulus heteroclitus) with asynchronous oocyte development and a repeat spawning reproductive strategy.\u003c/li\u003e\r\n\t\u003cli\u003eSpecies dependent differences in endocrine feedback responses, likely associated with different reproductive strategies, have been reported. Thus, the applicability domain may prove more restricted than currently assumed. In particular, the applicability to fish species with synchronous or group synchronous oocyte development patterns (see Wallace and Selman 1981) is unclear.\u003c/li\u003e\r\n\t\u003cli\u003eEuropean eel may be an exception to the generalizability of the negative feedback response to a non-aromatizable xenoandrogen (Huang et al. 1997).\u003c/li\u003e\r\n\t\u003cli\u003eReductions in plasma VTG concentrations and/or hepatic VTG mRNA abundance in females following exposure to 17\u0026beta;-trenbolone has been observed in Pimephales promelas, Oryzias latipes, Danio rerio, (Seki et al. 2006), Cyprinodon variegatus (Hemmer et al. 2008), Gambusia holbrooki and Gambusia affinis (Brockmeier et al. 2013)\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e[\u003cem\u003eAssessment provided by Ioanna Katsiadaki - reviewer\u003c/em\u003e]: \u0026nbsp;This is restricted clearly to female fish only as adversity is linked to reduced oestrogen synthesis (via reduced androgen synthesis); it is also limited to fully reproductive mature fish (not fish entering puberty or juvenile fish) and importantly is limited to fish that once they reach sexual maturity they spawn constantly. The latter is a reproductive strategy employed by fish that tend to occupy tropical areas (around the equator). Unfortunately most fish species have different reproductive strategies (annual life cycle) hence the level of gonadotropin expression (and consequently steroid production) is regulated by photoperiodic and temperature changes throughout the year. Even if a negative feedback mechanism operates in all of these species and in all life stages (which is certainly not the case) we still need to establish what is the relative strength of the AR agonist induced negative feedback to the environment-induced stimulation of gonadotropins! This link has never been studied and is critical if we really mean to protect wildlife.\u003c/p\u003e\r\n","key_event_essentiality":"\u003cul\u003e\r\n\t\u003cli\u003eIn general, few studies have directly addressed the essentiality of the proposed sequence of key events.\u003c/li\u003e\r\n\t\u003cli\u003eEkman et al. 2011 provide evidence that in fathead minnow, cessation of trenbolone exposure resulted in recovery of plasma E2 and VTG concentrations which were depressed by continuous exposure to 17beta trenbolone. This provides some support for the essentiality of these two key events.\u003c/li\u003e\r\n\t\u003cli\u003eEssentiality of the proposed negative feedback key event is supported by experimental work that evaluated the ability of AR agonists to reduce T or E2 production in vitro. There are no known reports of 17\u0026beta;-trenbolone directly inhibiting steroid biosynthesis. When tested in an in vitro steroidogenesis assay using H295R adrenal carcinoma cells, trenbolone caused a concentration-dependent increase in estradiol production, as opposed to any reductions in steroid hormone concentrations, an effect that was concurrent with increased transcription of CYP19 (aromatase) in the cell line (Gracia et al. 2007).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003e\u003cstrong\u003eBiological Plausibility\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThe biochemistry of steroidogenesis and the predominant role of the gonad in synthesis of the sex steroids are well established.\u003c/li\u003e\r\n\t\u003cli\u003eSimilarly, the role of E2 as the major regulator of hepatic vitellogenin production is widely documented in the literature.\u003c/li\u003e\r\n\t\u003cli\u003eThe direct link between reduced VTG concentrations in the plasma and reduced uptake into oocytes is highly plausible, as the plasma is the primary source of the VTG.\u003c/li\u003e\r\n\t\u003cli\u003eThe direct connection between reduced VTG uptake and impaired spawning/reduced cumulative fecundity is more tentative. It is not clear, for instance whether impaired VTG uptake limits oocyte growth and failure to reach a critical size in turn impairs physical or inter-cellular signaling processes that promote release of the oocyte from the surrounding follicles. In at least one experiment, oocytes with similar size to vitellogenic oocytes, but lacking histological staining characteristic of vitellogenic oocytes was observed (R. Johnson, personal communication). At present, the link between reductions in circulating VTG concentrations and reduced cumulative fecundity are best supported by the correlation between those endpoints across multiple experiments, including those that impact VTG via other molecular initiating events (Miller et al. 2007).\u003c/li\u003e\r\n\t\u003cli\u003eAt present, negative feedback is the most biologically plausible explanation for the reductions in ex vivo T and E2 production following exposure to 17\u0026beta;-trenbolone.There are no known reports of 17\u0026beta;-trenbolone directly inhibiting steroid biosynthesis. When tested in an in vitro steroidogenesis assay using H295R adrenal carcinoma cells, trenbolone caused a concentration-dependent increase in estradiol production, as opposed to any reductions in steroid hormone concentrations, an effect that was concurrent with increased transcription of CYP19 (aromatase) in the cell line (Gracia et al. 2007). Given the lack of any established direct effect on steroidogenic enzyme activity, negative feedback is currently the most likely explanation for the consistent effects observed in vivo. That said, many uncertainties regarding the exact mechanisms through which an exogenous, non-aromatizable, AR agonist elicits negative feedback remain.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConcordance of dose-response relationships: \u0026nbsp;\u003ca href=\"https://aopwiki.org/aops/23/pictures\"\u003eSee Concordance Table\u003c/a\u003e\u0026nbsp; \u003c/strong\u003e(available in Excel and PDF format)\u003c/p\u003e\r\n\r\n\u003cp\u003eThere are a limited number of studies in which multiple key events were considered in the same study following exposure to known, non-aromatizable, AR agonists. These were considered the most useful for evaluating the concordance of dose-response relationships. In general, effects on downstream key events occurred at concentrations equal to or greater than those at which upstream events occurred. For exposures to 17b-trenbolone, key events related to steroid production and circulating estradiol and vitellogenin concentrations were impacted at the same dose at which effects on cumulative fecundity were observed. Effects on vitellogenin transcription were only observed at greater concentrations, but data for comparable species and dose ranges were unavailable at present. For two other AR agonists tested in fish, available studies examined a single time-point only. Consequently, it was unclear whether lower effect concentrations for certain downstream KEs, relative to upstream were due to a lack of dose-response concordance, or due to decreased sensitivity of the upstream later in the exposure time-course.\u003c/p\u003e\r\n\r\n\u003cp\u003eWhile not directly addressing dose-response concordance, the dependence of the key events on the concentration of the androgen agonist has been established for all key events starting at and down-stream of reduced T synthesis. However, to date we are not aware of any studies that have established a concentration-response relationship between exposure to non-endogenous AR agonists (e.g., xenobiotics, pharmaceuticals) and circulating gonadotropin concentrations in fish or other vertebrates.\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eExposure of female fathead minnows to the AR agonist 17\u0026beta;-trenbolone for 21 d caused concentration-dependent reductions in circulating T, E2, and VTG concentrations over a range from 0.005 to 0.5 \u0026mu;g/L. The concentration response for all three variables had a \u0026ldquo;U\u0026rdquo;-shaped concentration response curve which may indicate concentration-dependent differences in the feedback response and/or compensatory processes. Histological evidence of reduced VTG uptake and reduced gonad stage were evident, although the concentration-response of histological effects was not determined. Despite the \u0026ldquo;U\u0026rdquo;-shaped concentration-response at the biochemical level, concentration-dependent reductions in cumulative fecundity were observed (Ankley et al. 2003). Effective concentrations were consistent with those causing phenotypic masculinization in female fish.\u003c/li\u003e\r\n\t\u003cli\u003eJensen et al. (2006) also demonstrated concentration-dependent reductions in circulating T, E2, and VTG following 21 d of in vivo exposure to 17\u0026alpha;-trenbolone (Jensen et al. 2006).\u003c/li\u003e\r\n\t\u003cli\u003eIn a time-course experiment in which female fathead minnows were exposed to to 33 or 472 ng 17\u0026beta;-trenbolone/L ex vivo T, ex vivo E2, plasma E2, and plasma VTG all showed concentration-dependent reductions that were consistent with the AOP (Ekman et al. 2011).\u003c/li\u003e\r\n\t\u003cli\u003eExposure of female fathead minnows to spironolactone, a pharmaceutical that binds the fathead minnow AR, for 21 d caused concentration-dependent reductions in cumulative fecundity, plasma VTG and VTG mRNA expression, and plasma E2 concentrations. The frequency and severity of females with decreased yolk accumulation, and increased oocyte atresia was concentration-dependent. The chemical also induced phenotypic masculinization in female fish. (Lalone et al. 2013).\u003c/li\u003e\r\n\t\u003cli\u003eExposure of female medaka to spironolactone caused concentration-dependent reductions in cumulative fecundity and VTG mRNA expression (impacts on steroid hormone concentrations were not measured). Spironolactone also caused phenotypic masculinization of female medaka (Lalone et al. 2013).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eTemporal concordance among the key events and adverse effect\u003c/strong\u003e: Temporal concordance between activation of the AR as a nuclear transcription factor and onset of a negative feedback response resulting in decreased gonadotropin secretion has not been established. Temporal concordance of the key events starting with reduced T biosynthesis and proceeding through reductions in plasma vitellogenin has been established (\u003ca href=\"https://aopwiki.org/aops/23/pictures\"\u003eConcordance Table\u003c/a\u003e). Temporal concordance beyond the key event of reductions in plasma vitellogenin has not been established, in large part due to disconnect in the time-scales over which the events can be measured. For example, most small fish used in reproductive toxicity testing can spawn anywhere from once daily to several days per week. Given the variability in daily spawning rates, it is neither practical nor effective to evaluate cumulative fecundity at a time scale shorter than roughly a week. Since the impacts at lower levels of biological organization can be detected within hours of exposure, lack of impact on cumulative fecundity before the other key events are impacted cannot be effectively measured. Overall, among those key events whose temporal concordance can reasonably be evaluated based on currently available data, the temporal profile observed is consistent with the AOP.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConsistency\u003c/strong\u003e: We are aware of no cases where the pattern of key events described was observed without also observing a significant impact on cumulative fecundity. Due to variability in the cumulative fecundity endpoint and potential compensatory responses ((Villeneuve et al. 2009; Villeneuve et al. 2013; Ankley et al. 2009b; Zhang et al. 2008; Ekman et al. 2012), the cumulative fecundity endpoint can be less sensitive than key events measured at lower levels of biological organization. Nonetheless, the occurrence of the final adverse outcome when the other key events are observed is very consistent. The final adverse effect is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events to reproductive dysfunction in small fish.\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eIn general, there is a consistent body of evidence linking exposure to an AR agonist to decreased T synthesis, E2 synthesis, circulating E2 and VTG concentrations, and cumulative fecundity in female fish. For example, the association between 17\u0026beta;-trenbolone exposure and reduced vitellogenin concentrations in females has been replicated in over a dozen independent experiments (Ekman et al. 2011; Ankley et al. 2003; Jensen et al. 2006; Ankley et al. 2010; Hemmer et al. 2008; Seki et al. 2006; Brockmeier et al. 2013). However, relatively few exogenous, non-aromatizable, AR agonists have been tested. Other than recent work with spironolactone (Lalone et al. 2013), we are not aware of the profile of responses being demonstrated for other AR agonists.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eUncertainties, inconsistencies, and data gaps\u003c/strong\u003e: There are three major areas of uncertainty and data gaps in the current AOP:\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eFirst, there remains considerable uncertainty as to the specific mechanism(s) through which AR agonism elicits a negative feedback response at the level of the hypothalamus and/or pituitary. There is also a substantial data gap relative to establishing that exposure to an AR agonist like 17\u0026beta;-trenbolone causes concentration-dependent reductions in circulating gonadotropins. That uncertainty is amplified further by the variation in feedback control along the endocrine axis for fish species employing different reproductive strategies. For example, gonadotropin regulation may be very different in species with synchronous oocyte maturation and annual or once per life-time reproductive strategies. Thus, there are considerable uncertainties related to the taxonomic relevance of this AOP to a broader range of fish species or other vertebrates.\u003c/li\u003e\r\n\t\u003cli\u003eThe second major uncertainty in this AOP relates to whether there is a direct biological linkage between impaired VTG uptake into oocytes and impaired spawning/reduced cumulative fecundity. Plausible biological connections have been hypothesized, but have not yet been tested experimentally.\u003c/li\u003e\r\n\t\u003cli\u003eA third uncertainty pertains to the chemical domain of applicability. In vivo, a number of chemicals that are detected as androgens in in vitro screening assays such as receptor binding assays or ligand-activated transcriptional assay can be aromatized to functional estrogens. Thus, in vivo such compounds may produce a profile of effects more consistent with estrogen receptor activation than AR activation or may produced mixed effects characteristic of either estrogen or androgen exposures (e.g., Pawlowski et al. 2004; Hornung et al. 2004). Examples of such aromatizable androgens include, testosterone, methyltestosterone, and androstenedione. Consequently, caution is warranted in applying this AOP based on in vitro screening data alone, without consideration for possible conversion to estrogens.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","quantitative_considerations":"\u003cp\u003e\u003cstrong\u003eAssessment of quantitative understanding of the AOP\u003c/strong\u003e: At present, the quantitative understanding of the AOP is insufficient to directly link a measure of chemical potency as an AR agonist (e.g., as measured in a transcriptional activation assay) to a predicted effect concentration at the level of cumulative fecundity. However, a number of mechanistic and statistical models are sufficiently developed to facilitate predictions of cumulative outcomes based on intermediate key event measurements such as circulating vitellogenin concentrations. Because the current models were developed based on a fairly limited range of model compounds and species, the general applicability and degree of accuracy and precision in the model-derived predictions remains uncertain.\u003c/p\u003e\r\n","optional_considerations":"\u003cp\u003eoptional\u003c/p\u003e\r\n","references":"\u003cul\u003e\r\n\t\u003cli\u003eAmano M, Iigo M, Ikuta K, Kitamura S, Yamada H, Yamamori K. 2000. Roles of melatonin in gonadal maturation of underyearling precocious male masu salmon. General and comparative endocrinology 120(2): 190-197.\u003c/li\u003e\r\n\t\u003cli\u003eAnkley GT, Bencic D, Cavallin JE, Jensen KM, Kahl MD, Makynen EA, et al. 2009. Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicol Sci 112(2): 344-353.\u003c/li\u003e\r\n\t\u003cli\u003eAnkley GT, Cavallin JE, Durhan EJ, Jensen KM, Kahl MD, Makynen EA, et al. 2012. A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic-pituitary-gonadal axis of fathead minnows. Aquatic toxicology 114-115: 88-95.\u003c/li\u003e\r\n\t\u003cli\u003eAnkley GT, Jensen KM, Durhan EJ, Makynen EA, Butterworth BC, Kahl MD, et al. 2005. Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas). Toxicol Sci 86(2): 300-308.\u003c/li\u003e\r\n\t\u003cli\u003eAnkley GT, Jensen KM, Kahl MD, Durhan EJ, Makynen EA, Cavallin JE, et al. 2010. Use of chemical mixtures to differentiate mechanisms of endocrine action in a small fish model. Aquatic toxicology 99(3): 389-396.\u003c/li\u003e\r\n\t\u003cli\u003eAnkley GT, Jensen KM, Kahl MD, Korte JJ, Makynen EA. 2001. Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ Toxicol Chem 20:1276-1290.\u003c/li\u003e\r\n\t\u003cli\u003eAnkley GT, Jensen KM, Kahl MD, Makynen EA, Blake LS, Greene KJ, et al. 2007. Ketoconazole in the fathead minnow (Pimephales promelas): reproductive toxicity and biological compensation. Environ Toxicol Chem 26(6): 1214-1223.\u003c/li\u003e\r\n\t\u003cli\u003eAnkley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, et al. 2003. Effects of the androgenic growth promoter 17-b-trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environmental Toxicology and Chemistry 22(6): 1350-1360.\u003c/li\u003e\r\n\t\u003cli\u003eAnkley GT, Kahl MD, Jensen KM, Hornung MW, Korte JJ, Makynen EA, et al. 2002. Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). Toxicological Sciences 67: 121-130.\u003c/li\u003e\r\n\t\u003cli\u003eAnkley GT, Miller DH, Jensen KM, Villeneuve DL, Martinovic D. 2008. Relationship of plasma sex steroid concentrations in female fathead minnows to reproductive success and population status. Aquatic toxicology 88(1): 69-74.\u003c/li\u003e\r\n\t\u003cli\u003eAraki N, Ohno K, Nakai M, Takeyoshi M, Iida M. 2005. Screening for androgen receptor activities in 253 industrial chemicals by in vitro reporter gene assays using AR-EcoScreen cells. Toxicology in vitro\u0026nbsp;: an international journal published in association with BIBRA 19(6): 831-842.\u003c/li\u003e\r\n\t\u003cli\u003eArukwe A, Goks\u0026oslash;yr A. 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comparative Hepatology 2(4): 1-21.\u003c/li\u003e\r\n\t\u003cli\u003eBaker ME. 1997. Steroid receptor phylogeny and vertebrate origins. Molecular and cellular endocrinology 135(2): 101-107.\u003c/li\u003e\r\n\t\u003cli\u003eBaker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.\u003c/li\u003e\r\n\t\u003cli\u003eBenninghoff AD, Thomas P. 2006. Gonadotropin regulation of testosterone production by primary cultured theca and granulosa cells of Atlantic croaker: I. Novel role of CaMKs and interactions between calcium- and adenylyl cyclase-dependent pathways. General and comparative endocrinology 147(3): 276-287.\u003c/li\u003e\r\n\t\u003cli\u003eBiales AD, Bencic DC, Lazorchak JL, Lattier DL. 2007. A quantitative real-time polymerase chain reaction method for the analysis of vitellogenin transcripts in model and nonmodel fish species. Environ Toxicol Chem 26(12): 2679-2686.\u003c/li\u003e\r\n\t\u003cli\u003eBohl CE, Chang C, Mohler ML, Chen J, Miller DD, Swaan PW, et al. 2004. A ligand-based approach to identify quantitative structure-activity relationships for the androgen receptor. Journal of medicinal chemistry 47(15): 3765-3776.\u003c/li\u003e\r\n\t\u003cli\u003eBowman CJ, Kroll KJ, Hemmer MJ, Folmar LC, Denslow ND. 2000. Estrogen-induced vitellogenin mRNA and protein in sheepshead minnow (Cyprinodon variegatus). General and comparative endocrinology 120(3): 300-313.\u003c/li\u003e\r\n\t\u003cli\u003eBreen MS, Villeneuve DL, Breen M, Ankley GT, Conolly RB. 2007. Mechanistic computational model of ovarian steroidogenesis to predict biochemical responses to endocrine active compounds. Annals of biomedical engineering 35(6): 970-981.\u003c/li\u003e\r\n\t\u003cli\u003eBrockmeier EK, Ogino Y, Iguchi T, Barber DS, Denslow ND. 2013. Effects of 17beta-trenbolone on Eastern and Western mosquitofish (Gambusia holbrooki and G. affinis) anal fin growth and gene expression patterns. Aquatic toxicology 128-129: 163-170.\u003c/li\u003e\r\n\t\u003cli\u003eCampbell BK, Baird DT, Webb R. 1998. Effects of dose of LH on androgen production and luteinization of ovine theca cells cultured in a serum-free system. Journal of reproduction and fertility 112(1): 69-77.\u003c/li\u003e\r\n\t\u003cli\u003eCentenera MM, Harris JM, Tilley WD, Butler LM. 2008. The contribution of different androgen receptor domains to receptor dimerization and signaling. Molecular endocrinology 22(11): 2373-2382.\u003c/li\u003e\r\n\t\u003cli\u003eCheng GF, Yuen CW, Ge W. 2007. Evidence for the existence of a local activin follistatin negative feedback loop in the goldfish pituitary and its regulation by activin and gonadal steroids. The Journal of endocrinology 195(3): 373-384.\u003c/li\u003e\r\n\t\u003cli\u003eClaessens F, Denayer S, Van Tilborgh N, Kerkhofs S, Helsen C, Haelens A. 2008. Diverse roles of androgen receptor (AR) domains in AR-mediated signaling. Nuclear receptor signaling 6: e008.\u003c/li\u003e\r\n\t\u003cli\u003eCutress ML, Whitaker HC, Mills IG, Stewart M, Neal DE. 2008. Structural basis for the nuclear import of the human androgen receptor. Journal of cell science 121(Pt 7): 957-968.\u003c/li\u003e\r\n\t\u003cli\u003eEick GN, Thornton JW. 2011. Evolution of steroid receptors from an estrogen-sensitive ancestral receptor. Molecular and cellular endocrinology 334(1-2): 31-38.\u003c/li\u003e\r\n\t\u003cli\u003eEkman DR, Hartig PC, Cardon M, Skelton DM, Teng Q, Durhan EJ, et al. 2012. Metabolite profiling and a transcriptional activation assay provide direct evidence of androgen receptor antagonism by bisphenol A in fish. Environmental science \u0026amp; technology 46(17): 9673-9680.\u003c/li\u003e\r\n\t\u003cli\u003eEkman DR, Villeneuve DL, Teng Q, Ralston-Hooper KJ, Martinovic-Weigelt D, Kahl MD, et al. 2011. Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17beta-trenbolone in fish. Environmental toxicology and chemistry / SETAC 30(2): 319-329.\u003c/li\u003e\r\n\t\u003cli\u003eEppig JJ. 1994. Further reflections on culture systems for the growth of oocytes in vitro. Human reproduction 9(6): 974-976.\u003c/li\u003e\r\n\t\u003cli\u003eGenovese G, Regueira M, Piazza Y, Towle DW, Maggese MC, Lo Nostro F. 2012. Time-course recovery of estrogen-responsive genes of a cichlid fish exposed to waterborne octylphenol. Aquatic toxicology 114-115: 1-13.\u003c/li\u003e\r\n\t\u003cli\u003eGopurappilly R, Ogawa S, Parhar IS. 2013. Functional significance of GnRH and kisspeptin, and their cognate receptors in teleost reproduction. Frontiers in endocrinology 4: 24.\u003c/li\u003e\r\n\t\u003cli\u003eGovoroun M, Chyb J, Breton B. 1998. Immunological cross-reactivity between rainbow trout GTH I and GTH II and their alpha and beta subunits: application to the development of specific radioimmunoassays. General and comparative endocrinology 111(1): 28-37.\u003c/li\u003e\r\n\t\u003cli\u003eGracia T, Hilscherova K, Jones PD, Newsted JL, Higley EB, Zhang X, et al. 2007. Modulation of steroidogenic gene expression and hormone production of H295R cells by pharmaceuticals and other environmentally active compounds. Toxicology and applied pharmacology 225(2): 142-153.\u003c/li\u003e\r\n\t\u003cli\u003eHabibi HR, Huggard DL. 1998. Testosterone regulation of gonadotropin production in goldfish. Comparative biochemistry and physiology Part C, Pharmacology, toxicology \u0026amp; endocrinology 119(3): 339-344.\u003c/li\u003e\r\n\t\u003cli\u003eHavelock JC, Rainey WE, Carr BR. 2004. Ovarian granulosa cell lines. Molecular and cellular endocrinology 228(1-2): 67-78.\u003c/li\u003e\r\n\t\u003cli\u003eHeemers HV, Tindall DJ. 2007. Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex. Endocrine reviews 28(7): 778-808.\u003c/li\u003e\r\n\t\u003cli\u003eHemmer MJ, Cripe GM, Hemmer BL, Goodman LR, Salinas KA, Fournie JW, et al. 2008. Comparison of estrogen-responsive plasma protein biomarkers and reproductive endpoints in sheepshead minnows exposed to 17beta-trenbolone. Aquatic toxicology 88(2): 128-136.\u003c/li\u003e\r\n\t\u003cli\u003eHong H, Fang H, Xie Q, Perkins R, Sheehan DM, Tong W. 2003. Comparative molecular field analysis (CoMFA) model using a large diverse set of natural, synthetic and environmental chemicals for binding to the androgen receptor. SAR and QSAR in environmental research 14(5-6): 373-388.\u003c/li\u003e\r\n\t\u003cli\u003eHornung MW, Jensen KM, Korte JJ, Kahl MD, Durhan EJ, Denny JS, Henry TR, Ankley GT. 2004. Mechanistic basis for estrogenic effects in fathead minnow (\u003cem\u003ePimephales promelas\u003c/em\u003e) following exposure to the androgen 17alpha-methyltestosterone: conversion of\u0026nbsp;17alpha-methytestosterone to 17alpha-methylestradiol. 66: 15-23.\u003c/li\u003e\r\n\t\u003cli\u003eIguchi T, Irie F, Urushitani H, Tooi O, Kawashima Y, Roberts M, et al. 2006. Availability of in vitro vitellogenin assay for screening of estrogenic and anti-estrogenic activities of environmental chemicals. Environ Sci 13(3): 161-183.\u003c/li\u003e\r\n\t\u003cli\u003eJamnongjit M, Hammes SR. 2005. Oocyte maturation: the coming of age of a germ cell. Seminars in reproductive medicine 23(3): 234-241.\u003c/li\u003e\r\n\t\u003cli\u003eJensen K, Korte J, Kahl M, Pasha M, Ankley G. 2001. Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). Comparative Biochemistry and Physiology Part C 128: 127-141.\u003c/li\u003e\r\n\t\u003cli\u003eJensen KM, Makynen EA, Kahl MD, Ankley GT. 2006. Effects of the feedlot contaminant 17alpha-trenbolone on reproductive endocrinology of the fathead minnow. Environmental science \u0026amp; technology 40(9): 3112-3117.\u003c/li\u003e\r\n\t\u003cli\u003eJolly C, Katsiadaki I, Le Belle N, Mayer I, Dufour S. 2006. Development of a stickleback kidney cell culture assay for the screening of androgenic and anti-androgenic endocrine disrupters. Aquatic toxicology 79(2): 158-166.\u003c/li\u003e\r\n\t\u003cli\u003eKah O, Pontet A, Nunez Rodriguez J, Calas A, Breton B. 1989. Development of an enzyme-linked immunosorbent assay for goldfish gonadotropin. Biology of reproduction 41(1): 68-73.\u003c/li\u003e\r\n\t\u003cli\u003eKim TS, Yoon CY, Jung KK, Kim SS, Kang IH, Baek JH, et al. 2010. In vitro study of Organization for Economic Co-operation and Development (OECD) endocrine disruptor screening and testing methods- establishment of a recombinant rat androgen receptor (rrAR) binding assay. The Journal of toxicological sciences 35(2): 239-243.\u003c/li\u003e\r\n\t\u003cli\u003eKorte JJ, Kahl MD, Jensen KM, Mumtaz SP, Parks LG, LeBlanc GA, et al. 2000. Fathead minnow vitellogenin: complementary DNA sequence and messenger RNA and protein expression after 17B-estradiol treatment. Environmental Toxicology and Chemistry 19(4): 972-981.\u003c/li\u003e\r\n\t\u003cli\u003eLalone CA, Villeneuve DL, Cavallin JE, Kahl MD, Durhan EJ, Makynen EA, et al. 2013. Cross species sensitivity to a novel androgen receptor agonist of environmental concern, spironolactone. Environ. Toxicol. Chem. 32: 2528-2541.\u003c/li\u003e\r\n\t\u003cli\u003eLeino R, Jensen K, Ankley G. 2005. Gonadal histology and characteristic histopathology associated with endocrine disruption in the adult fathead minnow. Environmental Toxicology and Pharmacology 19: 85-98.\u003c/li\u003e\r\n\t\u003cli\u003eLevavi-Sivan B, Bogerd J, Mananos EL, Gomez A, Lareyre JJ. 2010. Perspectives on fish gonadotropins and their receptors. General and comparative endocrinology 165(3): 412-437.\u003c/li\u003e\r\n\t\u003cli\u003eLi Z, Kroll KJ, Jensen KM, Villeneuve DL, Ankley GT, Brian JV, et al. 2011a. A computational model of the hypothalamic: pituitary: gonadal axis in female fathead minnows (Pimephales promelas) exposed to 17alpha-ethynylestradiol and 17beta-trenbolone. BMC systems biology 5: 63.\u003c/li\u003e\r\n\t\u003cli\u003eLi Z, Villeneuve DL, Jensen KM, Ankley GT, Watanabe KH. 2011b. A computational model for asynchronous oocyte growth dynamics in a batch-spawning fish. Can J Fish Aquat Sci 68: 1528-1538.\u003c/li\u003e\r\n\t\u003cli\u003eMagoffin DA. 2005. Ovarian theca cell. The international journal of biochemistry \u0026amp; cell biology 37(7): 1344-1349.\u003c/li\u003e\r\n\t\u003cli\u003eMak P, Cruz FD, Chen S. 1999. A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor. Environmental health perspectives 107(11): 855-860.\u003c/li\u003e\r\n\t\u003cli\u003eMarkov GV, Laudet V. 2011. Origin and evolution of the ligand-binding ability of nuclear receptors. Molecular and cellular endocrinology 334(1-2): 21-30.\u003c/li\u003e\r\n\t\u003cli\u003eMcMaster ME MK, Jardine JJ, Robinson RD, Van Der Kraak GJ. 1995. Protocol for measuring in vitro steroid production by fish gonadal tissue. Canadian Technical Report of Fisheries and Aquatic Sciences 1961 1961: 1-78.\u003c/li\u003e\r\n\t\u003cli\u003eMiller DH, Ankley GT. 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17b-trenbolone as a case study. Ecotoxicology and Environmental Safety 59: 1-9.\u003c/li\u003e\r\n\t\u003cli\u003eMiller DH, Jensen KM, Villeneuve DL, Kahl MD, Makynen EA, Durhan EJ, et al. 2007. Linkage of biochemical responses to population-level effects: a case study with vitellogenin in the fathead minnow (Pimephales promelas). Environ Toxicol Chem 26(3): 521-527.\u003c/li\u003e\r\n\t\u003cli\u003eMiller DH, Tietge JE, McMaster ME, Munkittrick KR, Xia X, Ankley GT. 2013. Assessment of Status of White Sucker (Catostomus Commersoni) Populations Exposed to Bleached Kraft Pulp Mill Effluent. Environmental toxicology and chemistry / SETAC.\u003c/li\u003e\r\n\t\u003cli\u003eMiller WL, Strauss JF, 3rd. 1999. Molecular pathology and mechanism of action of the steroidogenic acute regulatory protein, StAR. The Journal of steroid biochemistry and molecular biology 69(1-6): 131-141.\u003c/li\u003e\r\n\t\u003cli\u003eMiller WL. 1988. Molecular biology of steroid hormone synthesis. Endocrine reviews 9(3): 295-318.\u003c/li\u003e\r\n\t\u003cli\u003eMurphy CA, Rose KA, Rahman MS, Thomas P. 2009. Testing and applying a fish vitellogenesis model to evaluate laboratory and field biomarkers of endocrine disruption in Atlantic croaker (Micropogonias undulatus) exposed to hypoxia. Environmental toxicology and chemistry / SETAC 28(6): 1288-1303.\u003c/li\u003e\r\n\t\u003cli\u003eMurphy CA, Rose KA, Thomas P. 2005. Modeling vitellogenesis in female fish exposed to environmental stressors: predicting the effects of endocrine disturbance due to exposure to a PCB mixture and cadmium. Reproductive toxicology 19(3): 395-409.\u003c/li\u003e\r\n\t\u003cli\u003eNagahama Y, Yoshikumi M, Yamashita M, Sakai N, Tanaka M. 1993. Molecular endocrinology of oocyte growth and maturation in fish. Fish Physiology and Biochemistry 11: 3-14.\u003c/li\u003e\r\n\t\u003cli\u003eNavas JM, Segner H. 2006. Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances. Aquat Toxicol 80(1): 1-22.\u003c/li\u003e\r\n\t\u003cli\u003eNorris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press.\u003c/li\u003e\r\n\t\u003cli\u003eNorris JD, Joseph JD, Sherk AB, Juzumiene D, Turnbull PS, Rafferty SW, et al. 2009. Differential presentation of protein interaction surfaces on the androgen receptor defines the pharmacological actions of bound ligands. Chemistry \u0026amp; biology 16(4): 452-460.\u003c/li\u003e\r\n\t\u003cli\u003eOakley AE, Clifton DK, Steiner RA. 2009. Kisspeptin signaling in the brain. Endocrine reviews 30(6): 713-743.\u003c/li\u003e\r\n\t\u003cli\u003eOECD. 2012. Test No. 229: Fish Short Term Reproduction Assay. Paris, France:Organization for Economic Cooperation and Development.\u003c/li\u003e\r\n\t\u003cli\u003eOlsson P-E, Berg A, von Hofsten J, Grahn B, Hellqvist A, Larsson A, et al. 2005. Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone. Reproductive Biology and Endocrinology 3: 1-17.\u003c/li\u003e\r\n\t\u003cli\u003eOrganization for Economic Cooperation and Development. 2007. Report of Eight 21-day Fish Endocrine Screening Assays With Additional Test Substances for Phase-3 of the OECD Validation Program: Studies with Octylphenol in the Fathead Minnow (Pimephales promelas) and Zebrafish (Danio rerio) and with Sodium Pentachlorophenol and Androstenedione in the Fathead Minnow (Pimephales promelas). Phase-3 OECD 21-day Fish Screening Assay Validation Report Additional Test Substances Studies. Paris, France.\u003c/li\u003e\r\n\t\u003cli\u003ePawlowski S, Sauer A, Shears JA, Tyler CR, Braunbeck T. 2004. Androgenic and estrogenic effects of the synthetic androgen 17\u0026alpha;-methyltestosterone on sexual development and reproductive performance in the fathead minnow (Pimephales promelas) determined using the gonadal recrudescence assay. Aquat Toxicol 68:277-291.\u003c/li\u003e\r\n\t\u003cli\u003ePayne AH, Hales DB. 2004. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine reviews 25(6): 947-970.\u003c/li\u003e\r\n\t\u003cli\u003ePrat F, Sumpter JP, Tyler CR. 1996. Validation of radioimmunoassays for two salmon gonadotropins (GTH I and GTH II) and their plasma concentrations throughout the reproductivecycle in male and female rainbow trout (Oncorhynchus mykiss). Biology of reproduction 54(6): 1375-1382.\u003c/li\u003e\r\n\t\u003cli\u003ePrescott J, Coetzee GA. 2006. Molecular chaperones throughout the life cycle of the androgen receptor. Cancer letters 231(1): 12-19.\u003c/li\u003e\r\n\t\u003cli\u003eQuignot N, Bois FY. 2013. A computational model to predict rat ovarian steroid secretion from in vitro experiments with endocrine disruptors. PloS one 8(1): e53891.\u003c/li\u003e\r\n\t\u003cli\u003eSchmid T, Gonzalez-Valero J, Rufli H, Dietrich DR. 2002. Determination of vitellogenin kinetics in male fathead minnows (Pimephales promelas). Toxicol Lett 131(1-2): 65-74.\u003c/li\u003e\r\n\t\u003cli\u003eSchmieder P, Tapper M, Linnum A, Denny J, Kolanczyk R, Johnson R. 2000. Optimization of a precision-cut trout liver tissue slice assay as a screen for vitellogenin induction: comparison of slice incubation techniques. Aquat Toxicol 49(4): 251-268.\u003c/li\u003e\r\n\t\u003cli\u003eSchultz IR, Orner G, Merdink JL, Skillman A. 2001. Dose-response relationships and pharmacokinetics of vitellogenin in rainbow trout after intravascular administration of 17alpha-ethynylestradiol. Aquatic toxicology 51(3): 305-318.\u003c/li\u003e\r\n\t\u003cli\u003eSeki M, Fujishima S, Nozaka T, Maeda M, Kobayashi K. 2006. Comparison of response to 17 beta-estradiol and 17 beta-trenbolone among three small fish species. Environmental toxicology and chemistry / SETAC 25(10): 2742-2752.\u003c/li\u003e\r\n\t\u003cli\u003eSerafimova R, Walker J, Mekenyan O. 2002. Androgen receptor binding affinity of pesticide \u0026quot;active\u0026quot; formulation ingredients. QSAR evaluation by COREPA method. SAR and QSAR in environmental research 13(1): 127-134.\u003c/li\u003e\r\n\t\u003cli\u003eShoemaker JE, Gayen K, Garcia-Reyero N, Perkins EJ, Villeneuve DL, Liu L, et al. 2010. Fathead minnow steroidogenesis: in silico analyses reveals tradeoffs between nominal target efficacy and robustness to cross-talk. BMC systems biology 4: 89.\u003c/li\u003e\r\n\t\u003cli\u003eSkolness SY, Blanksma CA, Cavallin JE, Churchill JJ, Durhan EJ, Jensen KM, et al. 2013. Propiconazole Inhibits Steroidogenesis and Reproduction in the Fathead Minnow (Pimephales promelas). Toxicological sciences\u0026nbsp;: an official journal of the Society of Toxicology 132(2): 284-297.\u003c/li\u003e\r\n\t\u003cli\u003eSkolness SY, Durhan EJ, Garcia-Reyero N, Jensen KM, Kahl MD, Makynen EA, et al. 2011. Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas). Aquat Toxicol 103(3-4): 170-178.\u003c/li\u003e\r\n\t\u003cli\u003eSower SA, Freamat M, Kavanaugh SI. 2009. The origins of the vertebrate hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroid (HPT) endocrine systems: new insights from lampreys. General and comparative endocrinology 161(1): 20-29.\u003c/li\u003e\r\n\t\u003cli\u003eSperry TS, Thomas P. 1999. Identification of two nuclear androgen receptors in kelp bass (Paralabrax clathratus) and their binding affinities for xenobiotics: comparison with Atlantic croaker (Micropogonias undulatus) androgen receptors. Biology of reproduction 61(4): 1152-1161.\u003c/li\u003e\r\n\t\u003cli\u003eSun L, Wen L, Shao X, Qian H, Jin Y, Liu W, et al. 2010. Screening of chemicals with anti-estrogenic activity using in vitro and in vivo vitellogenin induction responses in zebrafish (Danio rerio). Chemosphere 78(7): 793-799.\u003c/li\u003e\r\n\t\u003cli\u003eSun L, Zha J, Spear PA, Wang Z. 2007. Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults. Comp Biochem Physiol C Toxicol Pharmacol 145(4): 533-541.\u003c/li\u003e\r\n\t\u003cli\u003eThornton JW. 2001. Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proceedings of the National Academy of Sciences of the United States of America 98(10): 5671-5676.\u003c/li\u003e\r\n\t\u003cli\u003eTilley WD, Marcelli M, Wilson JD, McPhaul MJ. 1989. Characterization and expression of a cDNA encoding the human androgen receptor. Proceedings of the National Academy of Sciences of the United States of America 86(1): 327-331.\u003c/li\u003e\r\n\t\u003cli\u003eTodorov M, Mombelli E, Ait-Aissa S, Mekenyan O. 2011. Androgen receptor binding affinity: a QSAR evaluation. SAR and QSAR in environmental research 22(3): 265-291.\u003c/li\u003e\r\n\t\u003cli\u003eTrudeau VL, Spanswick D, Fraser EJ, Larivi\u0026eacute;re K, Crump D, Chiu S, et al. 2000. The role of amino acid neurotransmitters in the regulation of pituitary gonadotropin release in fish. Biochemistry and Cell Biology 78: 241-259.\u003c/li\u003e\r\n\t\u003cli\u003eTrudeau VL. 1997. Neuroendocrine regulation of gonadotropin II release and gonadal growth in the goldfish, Carassius auratus. Reviews of Reproduction 2: 55-68.\u003c/li\u003e\r\n\t\u003cli\u003eTyler C, Sumpter J. 1996. Oocyte growth and development in teleosts. Reviews in Fish Biology and Fisheries 6: 287-318.\u003c/li\u003e\r\n\t\u003cli\u003eTyler C, van der Eerden B, Jobling S, Panter G, Sumpter J. 1996. Measurement of vitellogenin, a biomarker for exposure to oestrogenic chemicals, in a wide variety of cyprinid fish. Journal of Comparative Physiology and Biology 166: 418-426.\u003c/li\u003e\r\n\t\u003cli\u003evan der Burg B, Winter R, Man HY, Vangenechten C, Berckmans P, Weimer M, et al. 2010. Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds. Reproductive toxicology 30(1): 18-24.\u003c/li\u003e\r\n\t\u003cli\u003eVilleneuve DL, Ankley GT, Makynen EA, Blake LS, Greene KJ, Higley EB, et al. 2007. Comparison of fathead minnow ovary explant and H295R cell-based steroidogenesis assays for identifying endocrine-active chemicals. Ecotoxicol Environ Saf 68(1): 20-32.\u003c/li\u003e\r\n\t\u003cli\u003eVilleneuve DL, Breen M, Bencic DC, Cavallin JE, Jensen KM, Makynen EA, et al. 2013. Developing Predictive Approaches to Characterize Adaptive Responses of the Reproductive Endocrine Axis to Aromatase Inhibition: I. Data Generation in a Small Fish Model. Toxicological sciences\u0026nbsp;: an official journal of the Society of Toxicology.\u003c/li\u003e\r\n\t\u003cli\u003eVilleneuve DL, Mueller ND, Martinovic D, Makynen EA, Kahl MD, Jensen KM, et al. 2009. Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environ Health Perspect 117(4): 624-631.\u003c/li\u003e\r\n\t\u003cli\u003eWaller CL, Juma BW, Gray EJ, Kelce WR. 1996. Three-dimensional quantitative structure-activity relationships for androgen receptor ligands. Toxicology and Applied Pharmacolgy 137: 219-227.\u003c/li\u003e\r\n\t\u003cli\u003eWilson VS, Bobseine K, Lambright CR, Gray LE. 2002. A novel cell line, MDA-kb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists. Toxicological Sciences 66: 69-81.\u003c/li\u003e\r\n\t\u003cli\u003eWilson VS, Cardon MC, Gray LE, Jr., Hartig PC. 2007. Competitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human. Environmental toxicology and chemistry / SETAC 26(9): 1793-1802.\u003c/li\u003e\r\n\t\u003cli\u003eWolf JC, Dietrich DR, Friederich U, Caunter J, Brown AR. 2004. Qualitative and quantitative histomorphologic assessment of fathead minnow Pimephales promelas gonads as an endpoint for evaluating endocrine-active compounds: a pilot methodology study. Toxicol Pathol 32(5): 600-612.\u003c/li\u003e\r\n\t\u003cli\u003eYaron Z. 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129: 49-73.\u003c/li\u003e\r\n\t\u003cli\u003eYin D, He Y, Perera MA, Hong SS, Marhefka C, Stourman N, et al. 2003. Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor. Molecular pharmacology 63(1): 211-223.\u003c/li\u003e\r\n\t\u003cli\u003eYoung JM, McNeilly AS. 2010. Theca: the forgotten cell of the ovarian follicle. Reproduction 140(4): 489-504.\u003c/li\u003e\r\n\t\u003cli\u003eZhang X, Hecker M, Tompsett AR, Park JW, Jones PD, Newsted J, et al. 2008. Responses of the medaka HPG axis PCR array and reproduction to prochloraz and ketoconazole. Environ Sci Technol 42(17): 6762-6769.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","overall_assessment":"\u003cp\u003eAnnex 1 Table, Assessment of the relative level of confidence in the overall AOP based on rank ordered weight of evidence elements is attached in PDF format.\u003c/p\u003e\r\n","background":"\u003cp\u003eNo additional background\u003c/p\u003e\r\n","user_defined_mie":"25: Agonism, Androgen receptor","user_defined_ao":"360: Decrease, Population trajectory","oecd_project":"1.12","oecd_status_id":1,"graphical_representation_image_uid":"2016/11/29/aa7Androgen_receptor_agonism_leading_to_reproductive_dysfunction.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":"2017/03/20/3usvv7naq8_Annex1_for_AOP_23_AR_reproductive_dys_2017_03_20.pdf","changed_at":null,"development_strategy":null,"known_modulating_factors":null,"assigned_license_id":19,"handbook_id":1,"project_129":false},{"id":25,"title":"Aromatase inhibition leading to reproductive dysfunction","short_name":"Aromatase inhibition leading to reproductive dysfunction","corresponding_author_id":313,"abstract":"\u003cp\u003eThis adverse outcome pathway details the linkage between inhibition of gonadal aromatase activity in females and reproductive dysfunction, as measured through the adverse effect of reduced cumulative fecundity and spawning. Initial development of this AOP draws heavily on evidence collected using repeat-spawning fish species. Cumulative fecundity is the most apical endpoint considered in the OECD 229 Fish Short Term Reproduction Assay. The OECD 229 assay serves as screening assay for endocrine disruption and associated reproductive impairment (OECD 2012). Cumulative fecundity is one of several variables known to be of demographic significance in forecasting fish population trends. Therefore, this AOP has utility in supporting the application of measures of aromatase, or in silico predictions of the ability to inhibit aromatase, as a means to identify chemicals with known potential to adversely affect fish populations and potentially other oviparous vertebrates.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2024-04-01T16:34:44.000-04:00","status_id":1,"authors":"\u003cp\u003eDan Villeneuve, US EPA Mid-Continent Ecology Division (villeneuve.dan@epa.gov)\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cul\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eSex\u003c/strong\u003e: The AOP applies to females only. Males have relatively low gonadal aromatase expression and activity and the androgen 11-KT, rather than the estrogen E2 is a stronger driver of reproductive functions in males. That said, at least in fish, there is a potential autocrine and paracrine for estrogens synthesized in the brain in regulating reproductive behaviors. However, those potential effects are addressed through an alternative AOP that shares the MIE of aromatase inhibition.\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eLife stages\u003c/strong\u003e: The relevant life stages for this AOP are reproductively mature adults. This AOP does not apply to adult stages that lack a sexually mature ovary, for example as a result of seasonal or environmentally-induced gonadal senescence (i.e., through control of temperature, photo-period, etc. in a laboratory setting).\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eTaxonomic\u003c/strong\u003e: At present, the assumed taxonomic applicability domain of this AOP is class Osteichthyes. In all likelihood, the AOP will also prove applicable to all classes of fish (e.g., Agnatha and Chondrithyes as well). Additionally, all the key events described should be conserved among all oviparous vertebrates, suggesting that the AOP may also have relevance for amphibians, reptiles, and birds. However, species-specific differences in reproductive strategies/life histories, ADME (adsorption, distribution, metabolism, and elimination), compensatory reproductive endocrine responses may influence the outcomes, particularly from a quantitative standpoint.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","key_event_essentiality":"\u003cp\u003eSupport for the essentiality of a number of key events in the AOP was provided by several time-course, stop-reversibility, experiments with fathead minnows exposed to aromatase inhibitors.\u003c/p\u003e\r\n\r\n\u003cp\u003e1. Villeneuve et al. 2009 and 2013 examined a time-course of key event responses to fadrozole as well as the time-course of recovery following cessation of fadrozole delivery. Once fadrozole was removed from the system, ex vivo E2 production increased, followed by increases in plasma E2 concentrations, and then increases in plasma vitellogenin concentrations. Additionally, while exposure to the chemical was on-going, compensatory up-regulation of CYP19a1a gene expression resulted in increases in ex vivo E2 production, followed by increased plasma E2 and plasma VTG. The essentiality of aromatase inhibition relative to impaired E2 production was further supported by the observation of an \u0026quot;overshoot\u0026quot; in E2 production, relative to controls, shortly after cessation of fadrozole delivery.\u003c/p\u003e\r\n\r\n\u003cp\u003e2. Similar support was provided in a study by Ankley et al. (2009a). Cessation of prochloraz delivery resulted in rapid recovery of ex vivo E2 production and plasma E2 concentrations, with recovery of vitellogenin concentrations lagging slightly behind. Increased expression of cyp19a1a mRNA during the exposure period aligned with increased ex vivo E2 production, and increased plasma E2, compared to the first day of exposure.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nRationale for essentiality calls:\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026bull; Aromatase, inhibition: [Strong] There is good evidence from stop/reversibility studies that ceasing delivery of the aromatase inhibitor leads to recovery of the subsequent key events.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026bull; 17beta-estradiol synthesis by ovarian granulosa cells, reduction: [Strong] In both exposure studies and stop/reversibility studies, when ex vivo E2 production (as measure of this KE) recovers either through compensation or due to removal of the stressor, subsequent KEs have been shown to recover after a lag period.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026bull; plasma 17beta-estradiol concentrations, reduction: [Strong] In both exposure studies and stop/reversibility studies, when plasma E2 concentrations recover either through compensation or due to removal of the stressor, subsequent KEs have been shown to recover after a lag period.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026bull; vitellogenin production in liver (transcription, translation), reduction: [Moderate] This endpoint was not specifically examined in stop/reversibility studies with aromatase inhibitors, but biological plausibility provides strong support for the essentiality of this event.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026bull; plasma vitellogenin concentrations, reduction: [Strong] Shown to recover in a predictable fashion consistent with the order of events in the AOP in stop/recovery studies.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026bull; vitellogenin accumulation into oocytes and oocyte growth/development, reduction: [Weak] Some contradictory evidence regarding the essentiality of this event. No stop/reversibility studies have explicitly considered this key event.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026bull; cumulative fecundity and spawning, reductions: [Moderate] By definition, some degree of spawning is required to maintain population.\u003c/p\u003e\r\n","weight_of_evidence_summary":"\u003cp\u003e\u003cstrong\u003eBiological plausibility\u003c/strong\u003e: Biological plausibility refers to the structural or functional relationship between the key events based on our fundamental understanding of \u0026quot;normal biology\u0026quot;. In general, the biological plausibility and coherence linking aromatase inhibition through decreases in circulating concentrations of E2 is very solid. The biochemistry of steroidogenesis and the predominant role of the gonad in synthesis of the sex steroids is well established. Similarly, the role of E2 as the major regulator of hepatic vitellogenin production is widely documented in the literature. The direct link between reduced VTG concentrations in the plasma and reduced uptake into oocytes is highly plausible, as the plasma is the primary source of the VTG. However, the direct connection between reduced VTG uptake and impaired spawning/reduced cumulative fecundity is more tentative. It is not clear, for instance whether impaired VTG uptake limits oocyte growth and failure to reach a critical size in turn impairs physical or inter-cellular signaling processes that promote release of the oocyte from the surrounding follicles. In at least one experiment, oocytes with similar size to vitellogenic oocytes, but lacking histological staining characteristic of vitellogenic oocytes was observed (R. Johnson, personal communication). Regulation of oocyte maturation and spawning involves many factors other than vitellogenin accumulation (Clelland and Peng, 2009). At present, the link between reductions in circulating VTG concentrations and reduced cumulative fecundity are best supported by the correlation between those endpoints across multiple experiments, including those that impact VTG via other molecular initiating events (Miller et al. 2007).\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConcordance of dose-response relationships\u003c/strong\u003e: Dose response concordance considers the degree to which upstream events are shown to occur at test concentrations equal to or lower than those that cause significant effects on downstream key events, the underlying assumption being that all KEs can be measured with equal precision. There are a limited number of studies in which multiple key events were considered in the same study. These were considered the most useful for evaluating the concordance of dose-response relationships. In general, effects on downstream key events occurred at concentrations equal to or greater than those at which upstream events occurred (Concordance table: \u003ca class=\"external autonumber\" href=\"https://aopwiki.org/wiki/images/4/45/Aromatase_inhibition_dose-response_concordance_table_rev1.pdf\" rel=\"nofollow\" target=\"_blank\"\u003e[1]\u003c/a\u003e). However, there are exceptions. There are cases where no significant effects on estradiol synthesis by ovarian granulosa cells (ovary explants) were observed, but significant effects on plasma E2 or VTG concentrations were observed. Likewise, there are cases where impacts on plasma VTG were observed at concentrations lower than those reported to reduce plasma E2 concentrations. Based on knowledge of the studies in question, the apparent lack of concordance in some cases is driven by two primary factors. First, differences in the sensitivity and dynamic range of the measurements being made. Second, the effects of compensatory responses along the HPG axis. For instance, although ex vivo E2 production is rapidly affected by exposure to fadrozole, it is also a response that is more rapidly corrected through upregulation of aromatase transcripts (see Villeneuve et al. 2009), meaning that it recovers more quickly than plasma concentrations of E2 or plasma VTG concentrations. Thus, at certain time points, one can get an apparent effect on plasma E2 or T without a measurable impact on E2 production by the gonad tissue, because the upstream insult occurred earlier in time and was subsequently offset by a compensatory response, but the compensation has yet to propagate through the pathway. Sensitivity and dynamic range of the measurement methods is also an issue. Vitellogenin concentrations have a highly dynamic range and can change by orders of magnitude. Other endpoints like plasma steroids are regulated in a narrower range, making differences more difficult to distinguish statistically. Therefore, in our assessment, the deviations from concordance do not call the KERs into question.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eThe concentration-dependence of the key event responses with regard to the concentration of aromatase inhibitor has been established in vitro and/or in vivo for nearly all key events in the AOP.\u003c/p\u003e\r\n\r\n\u003col\u003e\r\n\t\u003cli\u003eConcentration-dependent aromatase inhibition: (Villeneuve et al. 2006; Ankley et al. 2005; M et al. 2004; AM et al. 2000; Shilling et al. 1999)\u003c/li\u003e\r\n\t\u003cli\u003eConcentration-dependent decreases in E2 production in vitro, ex vivo: (Ankley et al. 2002; Villeneuve et al. 2007; Villeneuve et al. 2009; Ankley et al. 2005; a Marca Pereira et al. 2011; Lee et al. 2006).\u003c/li\u003e\r\n\t\u003cli\u003eConcentration-dependent decreases in circulating E2 concentrations: (Ankley et al. 2002; Villeneuve et al. 2009; Ankley et al. 2005; Ankley et al. 2009a; GT et al. 2001)\u003c/li\u003e\r\n\t\u003cli\u003eConcentration-dependent decreases in vitellogenin mRNA expression: (Sun et al. 2010; Sun et al. 2011; Zhang et al. 2008)\u003c/li\u003e\r\n\t\u003cli\u003eConcentration-dependent decreases in circulating vitellogenin concentrations: (Ankley et al. 2002; Villeneuve et al. 2009; Ankley et al. 2005; Ankley et al. 2009a; Sun et al. 2007; GT et al. 2001; Ralston-Hooper et al. 2013)\u003c/li\u003e\r\n\t\u003cli\u003eConcentration-dependent reductions in VTG uptake into oocytes or impaired oocyte development: Concentration-dependence of these effects has not been well demonstrated. The effects, when seen, have typically been documented at the greatest exposure concentration tested, but concentration-dependence of the severity or frequency of the impact was not documented (e.g., (Ankley et al. 2002; Ankley et al. 2005; Sun et al. 2007)\u003c/li\u003e\r\n\t\u003cli\u003eConcentration-dependent reductions in cumulative fecundity: (Ankley et al. 2002; Ankley et al. 2005; Sun et al. 2007; Zhang et al. 2008)\u003c/li\u003e\r\n\t\u003cli\u003eDeclining population trajectory: Modeled population trajectories show a concentration-dependent reduction in projected population size, however, those results are driven by the concentration-dependence of cumulative fecundity. Population-level effects have not been measured directly.\u003c/li\u003e\r\n\u003c/ol\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\n\u003cstrong\u003eTemporal concordance\u003c/strong\u003e: Temporal concordance refers to the degree to which the data support the hypothesized sequence of the key events; i.e., the effect on KE1 is observed before the effect on KE2, which is observed before the effect on KE3 and so on. Temporal concordance of the AOP from aromatase inhibition to decreased E2 production, decreased circulating E2, and decreased plasma VTG concentrations has been established (e.g., (Villeneuve et al. 2009; Ankley et al. 2009a; Skolness et al. 2011). Temporal concordance has not been established beyond that key event, in large part due to disconnect in the time-scales over which the events can be measured. For example, most small fish used in reproductive toxicity testing will can spawn anywhere from once daily to several days per week. Given the variability in daily spawning rates, it is neither practical nor effective to evaluate cumulative fecundity at a time scale shorter than roughly a week. Since the impacts at lower levels of biological organization can be detected within hours of exposure, lack of impact on cumulative fecundity before the other key events are impacted cannot be effectively measured. Overall, among those key events whose temporal concordance can reasonably be evaluated, the temporal profile observed is consistent with the AOP.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eConsistency\u003c/strong\u003e: We are aware of no cases where the pattern of key events described was observed without also observing a significant impact on cumulative fecundity. The final adverse outcome is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events to reproductive dysfunction in small fish.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eUncertainties, inconsistencies, and data gaps\u003c/strong\u003e: The current major uncertainty in this AOP is whether there is a direct biological linkage between impaired VTG uptake into oocytes and impaired spawning/reduced cumulative fecundity. Plausible biological connections have been hypothesized, but have not yet been tested experimentally.\u003c/p\u003e\r\n","quantitative_considerations":"\u003cp\u003eAssessment of quantitative understanding of the AOP:\u003c/p\u003e\r\n\r\n\u003cp\u003eAt present, quantitative understanding of the AOP is approaching the point where an in vitro measurement of aromatase inhibition could be used as an input parameter into a series of coupled computational models that could generate quantitative predictions across multiple key events (e.g., circulating E2 concentrations, circulating VTG concentrations, predicted impacts on cumulative fecundity, and effects on population trajectories). A sequence of supporting models has been coupled together and predictions have been made for novel aromatase inhibitors (identified through high throughput in vitro screening; Conolly et al. 2017).\u003c/p\u003e\r\n\r\n\u003cp\u003eQ-AOP model-based predictions for short-term key events, were tested experimentally for five chemicals identified as aromatase inhibitors (Villeneuve et al. 2021).\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eQ-AOP-based predictions extending over the entire AOP were evaluated empirically for imazalil in 60 h, 10 d, and 21 day studies (Villeneuve et al. 2023).\u003c/p\u003e\r\n\r\n\u003cp\u003eIn general, model predictions were within 1-2 orders of magnitude, but differences and toxicokinetics and additional bioactivities (interactions in a broader AOP network) may lead to error/uncertainty in the model predictions.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","optional_considerations":"\u003cul\u003e\r\n\t\u003cli\u003eThe present AOP can provide potential support for the use of alternatives to the fish short term reproduction assay as a screen for aromatase inhibitors.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThe present AOP can serve as a foundation for tiered testing strategies and IATA related to risk assessments on chemicals identified as aromatase inhibitors.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThe present AOP can be used to guide endpoint selection for effects-based monitoring studies at sites where aromatase inhibition has been identified as a relevant biological activity of interest (e.g., through bioeffects prediction or bioeffects surveillance approaches; see Schroeder et al. 2016).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003eSchroeder, A. L., Ankley, G. T., Houck, K. A. and Villeneuve, D. L. (2016), Environmental surveillance and monitoring\u0026mdash;The next frontiers for high-throughput toxicology. Environ Toxicol Chem, 35: 513\u0026ndash;525. doi:10.1002/etc.3309\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eA series of computational models aligned with this AOP (i.e., a quantitative AOP construct) can be applied to estimate in vivo bench-mark doses based on in vitro screening results. Case studies evaluating this application are under way.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","references":"\u003cp\u003e1. OECD. 2012. Test No. 229: Fish Short Term Reproduction Assay. Paris, France:Organization for Economic Cooperation and Development.\u003c/p\u003e\r\n\r\n\u003cp\u003e2. Petkov PI, Temelkov S, Villeneuve DL, Ankley GT, Mekenyan OG. 2009. Mechanism-based categorization of aromatase inhibitors: a potential discovery and screening tool. SAR QSAR Environ Res 20(7-8): 657-678.\u003c/p\u003e\r\n\r\n\u003cp\u003e3. Lephart ED, Simpson ER. 1991. Assay of aromatase activity. Methods Enzymol 206: 477-483.\u003c/p\u003e\r\n\r\n\u003cp\u003e4. Letcher RJ, van Holsteijn I, Drenth H-J, Norstrom RJ, Bergman A, Safe S, et al. 1999. Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells. Toxicology and applied pharmacology 160: 10-20.\u003c/p\u003e\r\n\r\n\u003cp\u003e5. Sanderson J, Seinen W, Giesy J, van den Berg M. 2000. 2-chloro-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity. Toxicological Sciences 54: 121-127.\u003c/p\u003e\r\n\r\n\u003cp\u003e6. Villeneuve DL, Knoebl I, Kahl MD, Jensen KM, Hammermeister DE, Greene KJ, et al. 2006. Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas). Aquat Toxicol 76(3-4): 353-368.\u003c/p\u003e\r\n\r\n\u003cp\u003e7. Ankley GT, Kahl MD, Jensen KM, Hornung MW, Korte JJ, Makynen EA, et al. 2002. Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). Toxicological Sciences 67: 121-130.\u003c/p\u003e\r\n\r\n\u003cp\u003e8. Castro LF, Santos MM, Reis-Henriques MA. 2005. The genomic environment around the Aromatase gene: evolutionary insights. BMC evolutionary biology 5: 43.\u003c/p\u003e\r\n\r\n\u003cp\u003e9. Norris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press.\u003c/p\u003e\r\n\r\n\u003cp\u003e10. Yaron Z. 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129: 49-73.\u003c/p\u003e\r\n\r\n\u003cp\u003e11. Havelock JC, Rainey WE, Carr BR. 2004. Ovarian granulosa cell lines. Molecular and cellular endocrinology 228(1-2): 67-78.\u003c/p\u003e\r\n\r\n\u003cp\u003e12. Villeneuve DL, Ankley GT, Makynen EA, Blake LS, Greene KJ, Higley EB, et al. 2007. Comparison of fathead minnow ovary explant and H295R cell-based steroidogenesis assays for identifying endocrine-active chemicals. Ecotoxicol Environ Saf 68(1): 20-32.\u003c/p\u003e\r\n\r\n\u003cp\u003e13. McMaster ME MK, Jardine JJ, Robinson RD, Van Der Kraak GJ. 1995. Protocol for measuring in vitro steroid production by fish gonadal tissue. Canadian Technical Report of Fisheries and Aquatic Sciences 1961 1961: 1-78.\u003c/p\u003e\r\n\r\n\u003cp\u003e14. Ankley GT, Jensen KM, Kahl MD, Makynen EA, Blake LS, Greene KJ, et al. 2007. Ketoconazole in the fathead minnow (Pimephales promelas): reproductive toxicity and biological compensation. Environ Toxicol Chem 26(6): 1214-1223.\u003c/p\u003e\r\n\r\n\u003cp\u003e15. Villeneuve DL, Mueller ND, Martinovic D, Makynen EA, Kahl MD, Jensen KM, et al. 2009. Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environ Health Perspect 117(4): 624-631.\u003c/p\u003e\r\n\r\n\u003cp\u003e16. Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.\u003c/p\u003e\r\n\r\n\u003cp\u003e17. Jensen K, Korte J, Kahl M, Pasha M, Ankley G. 2001. Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). Comparative Biochemistry and Physiology Part C 128: 127-141.\u003c/p\u003e\r\n\r\n\u003cp\u003e18. Biales AD, Bencic DC, Lazorchak JL, Lattier DL. 2007. A quantitative real-time polymerase chain reaction method for the analysis of vitellogenin transcripts in model and nonmodel fish species. Environ Toxicol Chem 26(12): 2679-2686.\u003c/p\u003e\r\n\r\n\u003cp\u003e19. Schmieder P, Tapper M, Linnum A, Denny J, Kolanczyk R, Johnson R. 2000. Optimization of a precision-cut trout liver tissue slice assay as a screen for vitellogenin induction: comparison of slice incubation techniques. Aquat Toxicol 49(4): 251-268.\u003c/p\u003e\r\n\r\n\u003cp\u003e20. Navas JM, Segner H. 2006. Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances. Aquat Toxicol 80(1): 1-22.\u003c/p\u003e\r\n\r\n\u003cp\u003e21. Korte JJ, Kahl MD, Jensen KM, Mumtaz SP, Parks LG, LeBlanc GA, et al. 2000. Fathead minnow vitellogenin: complementary DNA sequence and messenger RNA and protein expression after 17B-estradiol treatment. Environmental Toxicology and Chemistry 19(4): 972-981.\u003c/p\u003e\r\n\r\n\u003cp\u003e22. Tyler C, van der Eerden B, Jobling S, Panter G, Sumpter J. 1996. Measurement of vitellogenin, a biomarker for exposure to oestrogenic chemicals, in a wide variety of cyprinid fish. Journal of Comparative Physiology and Biology 166: 418-426.\u003c/p\u003e\r\n\r\n\u003cp\u003e23. Tyler C, Sumpter J. 1996. Oocyte growth and development in teleosts. Reviews in Fish Biology and Fisheries 6: 287-318.\u003c/p\u003e\r\n\r\n\u003cp\u003e24. Leino R, Jensen K, Ankley G. 2005. Gonadal histology and characteristic histopathology associated with endocrine disruption in the adult fathead minnow. Environmental Toxicology and Pharmacology 19: 85-98.\u003c/p\u003e\r\n\r\n\u003cp\u003e25. Wolf JC, Dietrich DR, Friederich U, Caunter J, Brown AR. 2004. Qualitative and quantitative histomorphologic assessment of fathead minnow Pimephales promelas gonads as an endpoint for evaluating endocrine-active compounds: a pilot methodology study. Toxicol Pathol 32(5): 600-612.\u003c/p\u003e\r\n\r\n\u003cp\u003e26. Miller DH, Ankley GT. 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17b-trenbolone as a case study. Ecotoxicology and Environmental Safety 59: 1-9.\u003c/p\u003e\r\n\r\n\u003cp\u003e27. Ankley GT, Jensen KM, Durhan EJ, Makynen EA, Butterworth BC, Kahl MD, et al. 2005. Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas). Toxicol Sci 86(2): 300-308.\u003c/p\u003e\r\n\r\n\u003cp\u003e28. Ankley GT, Bencic D, Cavallin JE, Jensen KM, Kahl MD, Makynen EA, et al. 2009a. Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicol Sci 112(2): 344-353.\u003c/p\u003e\r\n\r\n\u003cp\u003e29. Skolness SY, Durhan EJ, Garcia-Reyero N, Jensen KM, Kahl MD, Makynen EA, et al. 2011. Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas). Aquat Toxicol 103(3-4): 170-178.\u003c/p\u003e\r\n\r\n\u003cp\u003e30. Breen M, Villeneuve DL, Ankley GT, Bencic DC, Breen MS, Watanabe KH, et al. 2013. Developing Predictive Approaches to Characterize Adaptive Responses of the Reproductive Endocrine Axis to Aromatase Inhibition: II. Computational Modeling. Toxicological sciences\u0026nbsp;: an official journal of the Society of Toxicology.\u003c/p\u003e\r\n\r\n\u003cp\u003e31. Breen MS, Villeneuve DL, Breen M, Ankley GT, Conolly RB. 2007. Mechanistic computational model of ovarian steroidogenesis to predict biochemical responses to endocrine active compounds. Annals of biomedical engineering 35(6): 970-981.\u003c/p\u003e\r\n\r\n\u003cp\u003e32. Shoemaker JE, Gayen K, Garcia-Reyero N, Perkins EJ, Villeneuve DL, Liu L, et al. 2010. Fathead minnow steroidogenesis: in silico analyses reveals tradeoffs between nominal target efficacy and robustness to cross-talk. BMC systems biology 4: 89.\u003c/p\u003e\r\n\r\n\u003cp\u003e33. Quignot N, Bois FY. 2013. A computational model to predict rat ovarian steroid secretion from in vitro experiments with endocrine disruptors. PloS one 8(1): e53891.\u003c/p\u003e\r\n\r\n\u003cp\u003e34. Ankley GT, Bencic DC, Cavallin JE, Jensen KM, Kahl MD, Makynen EA, et al. 2009b. Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicological sciences\u0026nbsp;: an official journal of the Society of Toxicology 112(2): 344-353.\u003c/p\u003e\r\n\r\n\u003cp\u003e35. Villeneuve DL, Breen M, Bencic DC, Cavallin JE, Jensen KM, Makynen EA, et al. 2013. Developing Predictive Approaches to Characterize Adaptive Responses of the Reproductive Endocrine Axis to Aromatase Inhibition: I. Data Generation in a Small Fish Model. Toxicological sciences\u0026nbsp;: an official journal of the Society of Toxicology.\u003c/p\u003e\r\n\r\n\u003cp\u003e36. Ankley GT, Cavallin JE, Durhan EJ, Jensen KM, Kahl MD, Makynen EA, et al. 2012. A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic-pituitary-gonadal axis of fathead minnows. Aquatic toxicology 114-115: 88-95.\u003c/p\u003e\r\n\r\n\u003cp\u003e37. Li Z, Kroll KJ, Jensen KM, Villeneuve DL, Ankley GT, Brian JV, et al. 2011a. A computational model of the hypothalamic: pituitary: gonadal axis in female fathead minnows (Pimephales promelas) exposed to 17alpha-ethynylestradiol and 17beta-trenbolone. BMC systems biology 5: 63.\u003c/p\u003e\r\n\r\n\u003cp\u003e38. A A, A G. 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comparative Hepatology 2(4): 1-21.\u003c/p\u003e\r\n\r\n\u003cp\u003e39. Sun L, Wen L, Shao X, Qian H, Jin Y, Liu W, et al. 2010. Screening of chemicals with anti-estrogenic activity using in vitro and in vivo vitellogenin induction responses in zebrafish (Danio rerio). Chemosphere 78(7): 793-799.\u003c/p\u003e\r\n\r\n\u003cp\u003e40. Iguchi T, Irie F, Urushitani H, Tooi O, Kawashima Y, Roberts M, et al. 2006. Availability of in vitro vitellogenin assay for screening of estrogenic and anti-estrogenic activities of environmental chemicals. Environ Sci 13(3): 161-183.\u003c/p\u003e\r\n\r\n\u003cp\u003e41. Murphy CA, Rose KA, Thomas P. 2005. Modeling vitellogenesis in female fish exposed to environmental stressors: predicting the effects of endocrine disturbance due to exposure to a PCB mixture and cadmium. Reproductive toxicology 19(3): 395-409.\u003c/p\u003e\r\n\r\n\u003cp\u003e42. Murphy CA, Rose KA, Rahman MS, Thomas P. 2009. Testing and applying a fish vitellogenesis model to evaluate laboratory and field biomarkers of endocrine disruption in Atlantic croaker (Micropogonias undulatus) exposed to hypoxia. Environmental toxicology and chemistry / SETAC 28(6): 1288-1303.\u003c/p\u003e\r\n\r\n\u003cp\u003e43. Ankley GT, Miller DH, Jensen KM, Villeneuve DL, Martinovic D. 2008. Relationship of plasma sex steroid concentrations in female fathead minnows to reproductive success and population status. Aquatic toxicology 88(1): 69-74.\u003c/p\u003e\r\n\r\n\u003cp\u003e44. Schmid T, Gonzalez-Valero J, Rufli H, Dietrich DR. 2002. Determination of vitellogenin kinetics in male fathead minnows (Pimephales promelas). Toxicol Lett 131(1-2): 65-74.\u003c/p\u003e\r\n\r\n\u003cp\u003e45. Schultz IR, Orner G, Merdink JL, Skillman A. 2001. Dose-response relationships and pharmacokinetics of vitellogenin in rainbow trout after intravascular administration of 17alpha-ethynylestradiol. Aquatic toxicology 51(3): 305-318.\u003c/p\u003e\r\n\r\n\u003cp\u003e46. Bowman CJ, Kroll KJ, Hemmer MJ, Folmar LC, Denslow ND. 2000. Estrogen-induced vitellogenin mRNA and protein in sheepshead minnow (Cyprinodon variegatus). General and comparative endocrinology 120(3): 300-313.\u003c/p\u003e\r\n\r\n\u003cp\u003e47. Genovese G, Regueira M, Piazza Y, Towle DW, Maggese MC, Lo Nostro F. 2012. Time-course recovery of estrogen-responsive genes of a cichlid fish exposed to waterborne octylphenol. Aquatic toxicology 114-115: 1-13.\u003c/p\u003e\r\n\r\n\u003cp\u003e48. Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, et al. 2003. Effects of the androgenic growth promoter 17-b-trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environmental Toxicology and Chemistry 22(6): 1350-1360.\u003c/p\u003e\r\n\r\n\u003cp\u003e49. Sun L, Zha J, Spear PA, Wang Z. 2007. Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults. Comp Biochem Physiol C Toxicol Pharmacol 145(4): 533-541.\u003c/p\u003e\r\n\r\n\u003cp\u003e50. Li Z, Villeneuve DL, Jensen KM, Ankley GT, Watanabe KH. 2011b. A computational model for asynchronous oocyte growth dynamics in a batch-spawning fish. Can J Fish Aquat Sci 68: 1528-1538.\u003c/p\u003e\r\n\r\n\u003cp\u003e51. Miller DH, Jensen KM, Villeneuve DL, Kahl MD, Makynen EA, Durhan EJ, et al. 2007. Linkage of biochemical responses to population-level effects: a case study with vitellogenin in the fathead minnow (Pimephales promelas). Environ Toxicol Chem 26(3): 521-527.\u003c/p\u003e\r\n\r\n\u003cp\u003e52. Miller DH, Tietge JE, McMaster ME, Munkittrick KR, Xia X, Ankley GT. 2013. Assessment of Status of White Sucker (Catostomus Commersoni) Populations Exposed to Bleached Kraft Pulp Mill Effluent. Environmental toxicology and chemistry / SETAC.\u003c/p\u003e\r\n\r\n\u003cp\u003e53. M H, M vdB, JT S. 2004. A comparison of human H295R and rat R2C cell lines as in vitro screening tools for effects on aromatase. Toxicol Lett 146: 183-194.\u003c/p\u003e\r\n\r\n\u003cp\u003e54. AM V, C H, V B, JC L. 2000. Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicology In Vitro 14: 227-234.\u003c/p\u003e\r\n\r\n\u003cp\u003e55. Shilling AD, Carlson DB, Williams DE. 1999. Rainbow trout, Oncorhynchus mykiss, as a model for aromatase inhibition. J Steroid Biochem Mol Biol 70(1-3): 89-95.\u003c/p\u003e\r\n\r\n\u003cp\u003e56. a Marca Pereira ML, Wheeler JR, Thorpe KL, Burkhardt-Holm P. 2011. Development of an ex vivo brown trout (Salmo trutta fario) gonad culture for assessing chemical effects on steroidogenesis. Aquat Toxicol 101(3-4): 500-511.\u003c/p\u003e\r\n\r\n\u003cp\u003e57. Lee PS, Pankhurst NW, King HR. 2006. Effects of aromatase inhibitors on in vitro steroidogenesis by Atlantic salmon (Salmo salar) gonadal and brain tissue. Comp Biochem Physiol A Mol Integr Physiol 145(2): 195-203.\u003c/p\u003e\r\n\r\n\u003cp\u003e58. GT A, KM J, MD K, JJ K, EA M. 2001. Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environmental Toxicology and Chemistry 20(6): 1276-1290.\u003c/p\u003e\r\n\r\n\u003cp\u003e59. Sun L, Shao X, Chi J, Hu X, Jin Y, Fu Z. 2011. Transcriptional responses in the brain, liver and gonad of Japanese ricefish (Oryzias latipes) exposed to two anti-estrogens. Comp Biochem Physiol C Toxicol Pharmacol 153(4): 392-401.\u003c/p\u003e\r\n\r\n\u003cp\u003e60. Zhang X, Hecker M, Tompsett AR, Park JW, Jones PD, Newsted J, et al. 2008. Responses of the medaka HPG axis PCR array and reproduction to prochloraz and ketoconazole. Environ Sci Technol 42(17): 6762-6769.\u003c/p\u003e\r\n\r\n\u003cp\u003e61. Ralston-Hooper KJ, Turner ME, Soderblom EJ, Villeneuve D, Ankley GT, Moseley MA, et al. 2013. Application of a Label-free, Gel-free Quantitative Proteomics Method for Ecotoxicological Studies of Small Fish Species. Environ Sci Technol 47(2): 1091-1100.\u003c/p\u003e\r\n\r\n\u003cp\u003e62. Clelland E, Peng C. Endocrine/paracrine control of zebrafish ovarian development. Mol Cell Endocrinol. 2009. 312(1-2):42-52. doi: 10.1016/j.mce.2009.04.009.\u003c/p\u003e\r\n\r\n\u003cp\u003e63.\u0026nbsp;Villeneuve DL, Blackwell BR, Blanksma CA, Cavallin JE, Cheng WY, Conolly RB, Conrow K, Feifarek DJ, Heinis LJ, Jensen KM, Kahl MD, Milsk RY, Poole ST, Randolph EC, Saari TW, Watanabe KH, Ankley GT. Case Study in 21st-Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Reproductive Outcomes in Fish In Vivo. Environ Toxicol Chem. 2023 Jan;42(1):100-116. doi: 10.1002/etc.5504.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e64.\u0026nbsp;Villeneuve DL, Blackwell BR, Cavallin JE, Cheng WY, Feifarek DJ, Jensen KM, Kahl MW, Milsk RY, Poole ST, Randolph EC, Saari TW, Ankley GT. Case Study in 21st Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Short-Term In Vivo Responses in Adult Female Fish. Environ Toxicol Chem. 2021 Apr;40(4):1155-1170. doi: 10.1002/etc.4968.\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e65.\u0026nbsp;Conolly RB, Ankley GT, Cheng W, Mayo ML, Miller DH, Perkins EJ, Villeneuve DL, Watanabe KH. Quantitative Adverse Outcome Pathways and Their Application to Predictive Toxicology. Environ Sci Technol. 2017 Apr 18;51(8):4661-4672. doi: 10.1021/acs.est.6b06230\u003c/p\u003e\r\n","overall_assessment":"","background":"","user_defined_mie":"36: Inhibition, Aromatase","user_defined_ao":"360: Decrease, Population trajectory","oecd_project":"1.12","oecd_status_id":1,"graphical_representation_image_uid":"2016/11/29/cc3Arom_inhib_reproductive_dysfunction_figure_Revised_2015-11-25.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2022-12-09T09:49:00.000-05:00","development_strategy":"","known_modulating_factors":"\u003cdiv\u003e\r\n\u003ctable class=\"table table-bordered table-fullwidth\"\u003e\r\n\t\u003cthead\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003cth\u003eModulating Factor (MF)\u003c/th\u003e\r\n\t\t\t\u003cth\u003eInfluence or Outcome\u003c/th\u003e\r\n\t\t\t\u003cth\u003eKER(s) involved\u003c/th\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/thead\u003e\r\n\t\u003ctbody\u003e\r\n\t\t\u003ctr\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\t\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\r\n\t\t\u003c/tr\u003e\r\n\t\u003c/tbody\u003e\r\n\u003c/table\u003e\r\n\u003c/div\u003e\r\n","assigned_license_id":20,"handbook_id":1,"project_129":false},{"id":26,"title":"Calcium-mediated neuronal ROS production and energy imbalance","short_name":"Calcium-mediated neuronal ROS production and energy imbalance","corresponding_author_id":56,"abstract":"\u003cp\u003eChemicals may lead to neurotoxicity through the inhibition of calcium ATPase activity, leading to increased intracellular calcium, increased ROS, and energy imbalance. This may lead to impaired nuerotransmission and oxidative neuronal damage.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2025-02-25T10:45:31.000-05:00","status_id":3,"authors":"","applicability_of_the_aop":"","key_event_essentiality":"","weight_of_evidence_summary":"","quantitative_considerations":"","optional_considerations":"","references":"","overall_assessment":"\u003cp\u003e\u003cem\u003eConsider the following criteria (may include references to KE Relationship pages): 1. concordance of dose-response relationships; 2. temporal concordance among the key events and adverse effect; 3. strength, consistency, and specificity of association of adverse effect and initiating event; 4. biological plausibility, coherence, and consistency of the experimental evidence; 5. alternative mechanisms that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP; 6. uncertainties, inconsistencies and data gaps. \u003c/em\u003e\u003c/p\u003e\r\n","background":"","user_defined_mie":"51: Inhibition, Ca++ ATPase","user_defined_ao":"356: Increased, Oxidative damage","oecd_project":"","oecd_status_id":null,"graphical_representation_image_uid":null,"saaop_status_id":3,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2025-02-25T10:45:31.000-05:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":21,"handbook_id":1,"project_129":false},{"id":27,"title":"Cholestatic Liver Injury induced by Inhibition of the Bile Salt Export Pump (ABCB11)","short_name":"Cholestatic Liver Injury induced by Inhibition of the Bile Salt Export Pump (ABCB11)","corresponding_author_id":242,"abstract":"\u003cp\u003eAdverse outcome pathways (AOPs) have been recently introduced in human risk assessment as pragmatic tools with multiple applications. As such, AOPs intend to provide a clear-cut mechanistic representation of pertinent toxicological effects. AOPs are typically composed of a molecular initiating event, a series of intermediate steps and key events, and an adverse outcome. In the current study, an AOP framework is proposed for cholestasis triggered by drug-mediated inhibition of the bile salt export pump transporter protein. For this purpose, an in-depth survey of relevant scientific literature was carried out in order to identify intermediate steps and key events. The latter include bile accumulation, the induction of oxidative stress and inflammation, and the activation of specific nuclear receptors. Collectively, these mechanisms drive both a deteriorative cellular response, which underlies directly caused cholestatic injury, and an adaptive cellular response, which is aimed at counteracting cholestatic insults. AOP development was performed according to OECD guidance, including critical consideration of the Bradford Hill criteria for weight of evidence assessment and the OECD key questions for evaluating AOP confidence. The postulated AOP is expected to serve as the basis for the development of new in vitro tests and the characterization of novel biomarkers of drug-induced cholestasis.\n\u003c/p\u003e","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2025-04-03T16:42:59.000-04:00","status_id":5,"authors":"\u003cp\u003eMathieu Vinken, Brigitte Landesmann, Marina Goumenou, Stefanie Vinken, Imran Shah, Hartmut Jaeschke, Catherine Willett, Maurice Whelan and Vera Rogiers.\n\u003c/p\u003e","applicability_of_the_aop":"","key_event_essentiality":"","weight_of_evidence_summary":"","quantitative_considerations":"","optional_considerations":"","references":"\u003cp\u003eAllen, K., Jaeschke, H., Copple, B.L. (2011). Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of inflammation during obstructive cholestasis. Am. J. Pathol. 178, 175-186.\n\u003c/p\u003e\u003cp\u003eAndersen, M.E., Clewell, R., Bhattacharya, S. (2012). Developing in vitro tools sufficient by themselves for 21st century risk assessment. In Towards the Replacement of In Vivo Repeated Dose Systemic Toxicity Testing Volume 2 (T. Gocht, M. Schwarz, Eds.), pp. 347-360. Imprimerie Mouzet, France.\n\u003c/p\u003e\u003cp\u003eAndersen, M.E., McMullen, P., Bhattacharya, S. (2013). Toxicogenomics for transcription factor-governed molecular pathways: moving on to roles beyond classification and prediction. Arch. Toxicol. 87, 7-11.\n\u003c/p\u003e\u003cp\u003eAnkley, G.T., Bennett, R.S., Erickson, R.J., Hoff, D.J., Hornung, M.W., Johnson, R.D., Mount, D.R., Nichols, J.W., Russom, C.L., Schmieder, P.K., Serrrano, J.A., Tietge, J.E., Villeneuve, D.L. (2010). Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. Environ. Toxicol. Chem. 29, 730-741.\n\u003c/p\u003e\u003cp\u003eBarbier, O., Torra, I.P., Sirvent, A., Claudel, T., Blanquart, C., Duran-Sandoval, D., Kuipers, F., Kosykh, V., Fruchart, J.C., Staels, B. (2003). FXR induces the UGT2B4 enzyme in hepatocytes: a potential mechanism of negative feedback control of FXR activity. Gastroenterology 124, 1926-1940.\n\u003c/p\u003e\u003cp\u003eBernstein, H., Bernstein, C., Payne, C.M., Dvorakova, K., Garewal, H. (2005). Bile acids as carcinogens in human gastrointestinal cancers. Mutat. Res. 589, 47-65.\n\u003c/p\u003e\u003cp\u003eBogdanffy, M.S., Daston, G., Faustman, E.M., Kimmel, C.A., Kimmel, G.L., Seed, J., Vu, V. (2001). Harmonization of cancer and noncancer risk assessment: proceedings of a consensus-building workshop. Toxicol. Sci. 61, 18-31.\n\u003c/p\u003e\u003cp\u003eBotla, R., Spivey, J.R., Aguilar, H., Bronk, S.F., Gores, G.J. (1995). Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism of UDCA cytoprotection. J. Pharmacol. Exp. Ther. 272, 930-938.\n\u003c/p\u003e\u003cp\u003eBoyer, J.L., Trauner, M., Mennone, A., Soroka, C.J., Cai, S.Y., Moustafa, T., Zollner, G., Lee, J.Y., Ballatori, N. (2006). Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents. Am. J. Physiol. Gastrointest. Liver Physiol. 290, G1124-G1130.\n\u003c/p\u003e\u003cp\u003eBoyer, L.B. (2009). Adaptive regulation of hepatocyte transporters in cholestasis. In The Liver: Biology and Pathobiology 5th edition (I.M. Arias, H.J. Alter, J.L. Boyer, D.E. Cohen, N. Fausto, D.A. Shafritz, A.W. Wolkoff, Eds.), pp. 681-702. Wiley-Blackwell, Oxford.\n\u003c/p\u003e\u003cp\u003eBjörnsson, E., Olsson, R. (2005). Outcome and prognostic markers in severe drug-induced liver disease. Hepatology 42, 481-489.\n\u003c/p\u003e\u003cp\u003eBlomme, E.A., Yang, Y., Waring, J.F. (2009). Use of toxicogenomics to understand mechanisms of drug-induced hepatotoxicity during drug discovery and development. Toxicol. Lett. 186, 22-31.\n\u003c/p\u003e\u003cp\u003eChen, H.L., Chen, H.L., Liu, Y.J., Feng, C.H., Wu, C.Y., Shyu, M.K., Yuan, R.H., Chang, M.H. (2005). Developmental expression of canalicular transporter genes in human liver. J. Hepatol. 43, 472-477.\n\u003c/p\u003e\u003cp\u003eCheng, X., Buckley, D., Klaassen, C.D. (2007). Regulation of hepatic bile acid transporters Ntcp and Bsep expression. Biochem. Pharmacol. 74, 1665-1676.\n\u003c/p\u003e\u003cp\u003eCherrington, N.J., Slitt, A.L., Li, N., Klaassen, C.D. (2004). Lipopolysaccharide-mediated regulation of hepatic transporter mRNA levels in rats. Drug Metab. Dispos. 32, 734-741.\n\u003c/p\u003e\u003cp\u003eDawson, S., Stahl, S., Paul, N., Barber, J., Kenna, J.G. (2011). In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab. Dispos. 40, 130-138.\n\u003c/p\u003e\u003cp\u003eDenson, L.A., Sturm, E., Echevarria, W., Zimmerman, T.L., Makishima, M., Mangelsdorf, D.J., Karpen, S.J. (2001). The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 121, 140-147.\n\u003c/p\u003e\u003cp\u003eEchchgadda, I., Song, C.S., Oh, T., Ahmed, M., De La Cruz, I.J., Chatterjee, B. (2007). The xenobiotic-sensing nuclear receptors pregnane X receptor, constitutive androstane receptor, and orphan nuclear receptor hepatocyte nuclear factor 4alpha in the regulation of human steroid-/bile acid-sulfotransferase. Mol. Endocrinol. 21, 2099-2111.\n\u003c/p\u003e\u003cp\u003eEloranta, J.J., Kullak-Ublick, G.A. (2005). Coordinate transcriptional regulation of bile acid homeostasis and drug metabolism. Arch. Biochem. Biophys. 433, 397-412.\n\u003c/p\u003e\u003cp\u003eEU. (2003). Directive 2003/15/EC of the European parliament, of the council of 27 February amending council directive 76/768/EEC on the approximation of the laws of the member states relating to cosmetic products. OV. J. L. 066, 26-35.\n\u003c/p\u003e\u003cp\u003eFattinger, K., Funk, C., Pantze, M., Weber, C., Reichen, J., Stieger, B., Meier, P.J. (2001). The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin. Pharmacol. Ther. 69, 223-231.\n\u003c/p\u003e\u003cp\u003eFaucette, S.R., Sueyoshi, T., Smith, C.M., Negishi, M., Lecluyse, E.L., Wang, H. (2006). Differential regulation of hepatic CYP2B6 and CYP3A4 genes by constitutive androstane receptor but not pregnane X receptor. J. Pharmacol. Exp. Ther. 317, 1200-1209.\n\u003c/p\u003e\u003cp\u003eFeingold, K.R., Spady, D.K., Pollock, A.S., Moser, A.H., Grunfeld, C. (1996). Endotoxin, TNF, and IL-1 decrease cholesterol 7 alpha-hydroxylase mRNA levels and activity. J. Lipid Res. 37, 223-228.\n\u003c/p\u003e\u003cp\u003eFunk, C., Pantze, M., Jehle, L., Ponelle, C., Scheuermann, G., Lazendic, M., Gasser, R. (2001). Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats: correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate. Toxicology 167, 83-98.\n\u003c/p\u003e\u003cp\u003eGnerre, C., Blättler, S., Kaufmann, M.R., Looser, R., Meyer, U.A. (2004). Regulation of CYP3A4 by the bile acid receptor FXR: evidence for functional binding sites in the CYP3A4 gene. Pharmacogenetics 14, 635-645.\n\u003c/p\u003e\u003cp\u003eGollamudi, R., Gupta, D., Goel, S., Mani, S. (2008). Novel orphan nuclear receptors-coregulator interactions controlling anti-cancer drug metabolism. Curr. Drug Metab. 9, 611-613.\n\u003c/p\u003e\u003cp\u003eGoodwin, B., Jones, S.A., Price, R.R., Watson, M.A., McKee, D.D., Moore, L.B., Galardi, C., Wilson, J.G., Lewis, M.C., Roth, M.E., Maloney, P.R., Willson, T.M., Kliewer, S.A. (2000). A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol. Cell 6, 517-526.\n\u003c/p\u003e\u003cp\u003eGores, G.J., Miyoshi, H., Botla, R., Aguilar, H.I., Bronk, S.F. (1998). Induction of the mitochondrial permeability transition as a mechanism of liver injury during cholestasis: a potential role for mitochondrial proteases. Biochim. Biophys. Acta 1366, 167-175.\n\u003c/p\u003e\u003cp\u003eGreen, R.M., Hoda, F., Ward, K.L. (2000). Molecular cloning and characterization of the murine bile salt export pump. Gene 241, 117-123.\n\u003c/p\u003e\u003cp\u003eGu, X., Ke S., Liu, D., Sheng, T., Thomas, P.E., Rabson, A.B., Gallo, M.A., Xie ,W., Tian, Y. (2006). Role of NF-kappaB in regulation of PXR-mediated gene expression: a mechanism for the suppression of cytochrome P-450 3A4 by proinflammatory agents. J. Biol. Chem. 281, 17882-17889.\n\u003c/p\u003e\u003cp\u003eGujral, J.S., Farhood, A., Bajt, M.L., Jaeschke, H. (2003). Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice. Hepatology 38, 355-363.\n\u003c/p\u003e\u003cp\u003eGujral, J.S., Liu, J., Farhood, A., Hinson, J.A., Jaeschke, H. (2004). Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice. Am. J. Physiol. Gastrointest. Liver Physiol. 286, G499-G507.\n\u003c/p\u003e\u003cp\u003eHill, A.B. (1965). The environment and disease: association or causation? Proc. R. Soc. Med. 58, 295-300.\n\u003c/p\u003e\u003cp\u003eHofmann, A.F. (2009). Bile acids and the enterohepatic circulation. In The Liver: Biology and Pathobiology (I.M.; Arias, H.J., Alter, J.L., Boyer, D.E., Cohen, N., Fausto, D.A., Shafritz, A.W., Wolkoff Eds.), pp. 289-304. Wiley-Blackwell, Oxford.\n\u003c/p\u003e\u003cp\u003e\u003ca rel=\"nofollow\" target=\"_blank\" class=\"external free\" href=\"http://caat.jhsph.edu/\"\u003ehttp://caat.jhsph.edu/\u003c/a\u003e (consulted June 2013).\n\u003c/p\u003e\u003cp\u003e\u003ca rel=\"nofollow\" target=\"_blank\" class=\"external free\" href=\"http://www.seurat-1.eu/\"\u003ehttp://www.seurat-1.eu/\u003c/a\u003e (consulted June 2013).\n\u003c/p\u003e\u003cp\u003eJaeschke, H., Hasegawa, T. (2006). Role of neutrophils in acute inflammatory liver injury. Liver Int. 26, 912-919.\n\u003c/p\u003e\u003cp\u003eJemnitz, K., Veres, Z., Vereczkey, L. (2010). Contribution of high basolateral bile salt efflux to the lack of hepatotoxicity in rat in response to drugs inducing cholestasis in human. Toxicol. Sci. 115, 80-88.\n\u003c/p\u003e\u003cp\u003eJulien, E., Boobis, A.R., Olin, S.S. (2009). The key events dose-response framework: a cross-disciplinary mode-of-action based approach to examining dose-response and thresholds. Crit. Rev. Food Sci. Nutr. 49, 682-689.\n\u003c/p\u003e\u003cp\u003eJung, D., Mangelsdorf, D.J., Meyer, U.A. (2006). Pregnane X receptor is a target of farnesoid X receptor. J. Biol. Chem. 281, 19081-19091.\n\u003c/p\u003e\u003cp\u003eJung, D., Elferink, M.G., Stellaard, F., Groothuis, G.M. (2007). Analysis of bile acid-induced regulation of FXR target genes in human liver slices. Liver Int. 27, 137-144.\n\u003c/p\u003e\u003cp\u003eKast, H.R., Goodwin, B., Tarr, P.T., Jones, S.A., Anisfeld, A.M., Stoltz, C.M., Tontonoz, P., Kliewer, S., Willson, T.M., Edwards, P.A. (2002). Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J. Biol. Chem. 277, 2908-2915. \n\u003c/p\u003e\u003cp\u003eKim, M.S., Shigenaga, J., Moser, A., Grunfeld, C., Feingold, K.R. (2004). Suppression of DHEA sulfotransferase (Sult2A1) during the acute-phase response. Am. J. Physiol. Endocrinol. Metab. 287, E731-E738.\n\u003c/p\u003e\u003cp\u003eKis, E., Ioja, E., Rajnai, Z., Jani, M., Méhn, D., Herédi-Szabó, K., Krajcsi, P. (2012). BSEP inhibition: in vitro screens to assess cholestatic potential of drugs. Toxicol. In Vitro 26, 1294-1299.\n\u003c/p\u003e\u003cp\u003eKnisely, A.S., Strautnieks, S.S., Meier, Y., Stieger, B., Byrne, J.A., Portmann, B.C., Bull, L.N., Pawlikowska, L., Bilezikçi, B., Ozçay, F., László, A., Tiszlavicz, L., Moore, L., Raftos, J., Arnell, H., Fischler, B., Németh, A., Papadogiannakis, N., Cielecka-Kuszyk, J., Jankowska, I. (2006). Hepatocellular carcinoma in ten children under five years of age with bile salt export pump deficiency. Hepatology 44, 478-486.\n\u003c/p\u003e\u003cp\u003eKocarek, T.A., Shenoy, S.D., Mercer-Haines, N.A., Runge-Morris, M. (2002). Use of dominant negative nuclear receptors to study xenobiotic-inducible gene expression in primary cultured hepatocytes. J. Pharmacol. Toxicol. Methods 47, 177-187.\n\u003c/p\u003e\u003cp\u003eKostrubsky, V.E., Vore, M., Kindt, E., Burliegh, J., Rogers, K., Peter, G., Altrogge, D., Sinz, M.W. (2001). The effect of troglitazone biliary excretion on metabolite distribution and cholestasis in transporter-deficient rats. Drug Metab. Dispos. 29, 1561-1566.\n\u003c/p\u003e\u003cp\u003eKrasowski, M.D., Yasuda, K., Hagey, L.R., Schuetz, E.G. (2005). Evolution of the pregnane X receptor: adaptation to cross-species differences in biliary bile salts. Mol. Endocrinol. 19, 1720-1739.\n\u003c/p\u003e\u003cp\u003eKuntz, E., Kuntz, H.D. (2008). Cholestasis. In Hepatology: Textbook and Atlas\u0026#160;: History, Morphology, Biochemistry, Diagnostics, Clinic, Therapy 3rd edition (E. Kuntz E., H.-D. Kuntz Eds.), pp. 235-250. Springer, Heidelberg.\n\u003c/p\u003e\u003cp\u003eLandesmann, B., Goumenou, M., Munn, S., Whelan, M. (2012). Description of prototype modes-of-action related to repeated dose toxicity. JRC Scientific and Policy Report 75689.\n\u003c/p\u003e\u003cp\u003eLang, C., Meier, Y., Stieger, B., Beuers, U., Lang, T., Kerb, R., Kullak-Ublick, G.A., Meier, P.J., Pauli-Magnus, C. (2007). Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury. Pharmacogenet. Genomics 17, 47-60.\n\u003c/p\u003e\u003cp\u003eLang, T., Haberl, M., Jung, D., Drescher, A., Schlagenhaufer, R., Keil, A., Mornhinweg, E., Stieger, B., Kullak-Ublick, G.A., Kerb, R. (2006). Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11). Drug Metab. Dispos. 34, 1582-1599.\n\u003c/p\u003e\u003cp\u003eLecureur, V., Sun, D., Hargrove, P., Schuetz, E.G., Kim, R.B., Lan, L.B., Schuetz, J.D. (2000). Cloning and expression of murine sister of P-glycoprotein reveals a more discriminating transporter than MDR1/P-glycoprotein. Mol. Pharmacol. 57, 24-35.\n\u003c/p\u003e\u003cp\u003eLee, J., Azzaroli, F., Wang, L., Soroka, C.J., Gigliozzi, A., Setchell, K.D., Kramer, W., Boyer, J.L. (2001). Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat. Gastroenterology 121, 1473-1484.\n\u003c/p\u003e\u003cp\u003eLi-Masters, T., Morgan, E.T. (2001). Effects of bacterial lipopolysaccharide on phenobarbital-induced CYP2B expression in mice. Drug Metab. Dispos. 29, 252-257.\n\u003c/p\u003e\u003cp\u003eLucena, M.I., Andrade, R.J., Kaplowitz, N., García-Cortes, M., Fernández, M.C., Romero-Gomez, M., Bruguera, M., Hallal, H,. Robles-Diaz, M., Rodriguez-González, J.F., Navarro, J.M., Salmeron, J., Martinez-Odriozola, P., Pérez-Alvarez, R., Borraz, Y., Hidalgo, R. (2009). Phenotypic characterization of idiosyncratic drug-induced liver injury: the influence of age and sex. Hepatology 49, 2001-2009.\n\u003c/p\u003e\u003cp\u003eMaher, J.M., Cheng, X., Slitt, A.L., Dieter, M.Z., Klaassen, C.D. (2005). Induction of the multidrug resistance-associated protein family of transporters by chemical activators of receptor-mediated pathways in mouse liver. Drug Metab. Dispos. 33, 956-962. \n\u003c/p\u003e\u003cp\u003eMazzon, E., Cuzzocrea, S. (2003). Role of iNOS in hepatocyte tight junction alteration in mouse model of experimental colitis. Cell. Mol. Biol. 49, 45-57.\n\u003c/p\u003e\u003cp\u003eMeek, M.E., Bucher, J.R., Cohen, S.M., Dellarco, V., Hill, R.N., Lehman-McKeeman, L.D., Longfellow, D.G., Pastoor, T., Seed, J., Patton, D.E. (2003). A framework for human relevance analysis of information on carcinogenic modes of action. Crit. Rev. Toxicol. 33, 591-653.\n\u003c/p\u003e\u003cp\u003eMeier, Y., Pauli-Magnus, C., Zanger, U.M., Klein, K., Schaeffeler, E., Nussler, A.K., Nussler, N., Eichelbaum, M., Meier, P.J., Stieger, B. (2006). Interindividual variability of canalicular ATP-binding-cassette (ABC)-transporter expression in human liver. Hepatology 44, 62-74.\n\u003c/p\u003e\u003cp\u003eMennone, A., Soroka, C.J., Harry, K.M., Boyer, J.L. (2010). Role of breast cancer resistance protein in the adaptive response to cholestasis. Drug Metab. Dispos. 38, 1673-1678. \n\u003c/p\u003e\u003cp\u003eMorgan, R.E., Trauner, M., van Staden, C.J., Lee, P.H., Ramachandran, B., Eschenberg, M., Afshari, C.A., Qualls, C.W. Jr., Lightfoot-Dunn, R., Hamadeh, H.K. (2010). Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol. Sci. 118, 485-500.\n\u003c/p\u003e\u003cp\u003eNRC. (2007). Toxicity testing in the 21st century: a vision and a strategy. The National Academies Press, Washington DC.\n\u003c/p\u003e\u003cp\u003eOECD. (2011). Report of the workshop on using mechanistic information on forming chemical categories. Series on testing and assessment No. 138.\n\u003c/p\u003e\u003cp\u003eOECD. (2012a). Proposal for a template and guidance on developing and assessing the completeness of adverse outcome pathways. \n\u003c/p\u003e\u003cp\u003eOECD. (2012b). The adverse outcome pathway for skin sensitization initiated by covalent binding to proteins part I: scientific evidence. Series on testing and assessment No. 168.\n\u003c/p\u003e\u003cp\u003eOude Elferink, R.P., Paulusma, C.C., Groen, A.K. (2006). Hepatocanalicular transport defects: pathophysiologic mechanisms of rare diseases. Gastroenterology 13, 908-925.\n\u003c/p\u003e\u003cp\u003eOzer, J., Ratner, M., Shaw, M., Bailey, W., Schomaker, S. (2008). The current state of serum biomarkers of hepatotoxicity. Toxicology 245, 194-205.\n\u003c/p\u003e\u003cp\u003ePadda, M.S., Sanchez, M., Akhtar, A.J., Boyer, J.L. (2011). Drug-induced cholestasis. Hepatology 53, 1377-1387.\n\u003c/p\u003e\u003cp\u003ePagani, R., Portolés, M.T., De La Viña, S., Melzner, I., Vergani, G. (2003). Alterations induced on cytoskeleton by Escherichia coli endotoxin in different types of rat liver cell cultures. Histol. Histopathol. 1, 837-848.\n\u003c/p\u003e\u003cp\u003ePauli-Magnus, C., Meier, P.J. (2006). Hepatobiliary transporters and drug-induced cholestasis. Hepatology 44, 778-787.\n\u003c/p\u003e\u003cp\u003ePauwels, M., Rogiers, V. (2010). Human health safety evaluation of cosmetics in the EU: a legally imposed challenge to science. Toxicol. Appl. Pharmacol. 243, 260-274.\n\u003c/p\u003e\u003cp\u003eRoman, I.D., Monte, M.J., Gonzalez-Buitrago, J.M., Esteller, A., Jimenez, R. (1990). Inhibition of hepatocytary vesicular transport by cyclosporin A in the rat: relationship with cholestasis and hyperbilirubinemia. Hepatology 12, 83-91.\n\u003c/p\u003e\u003cp\u003eSalgia, R., Becker, J.H., Sayeed, M.M. (1993). Altered membrane fluidity in rat hepatocytes during endotoxic shock. Mol. Cell. Biochem. 121, 143-148.\n\u003c/p\u003e\u003cp\u003eSchoemaker, M.H., Conde de la Rosa, L., Buist-Homan, M., Vrenken, T.E., Havinga, R., Poelstra, K., Haisma, H.J., Jansen, P.L., Moshage, H. (2004). Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways. Hepatology 39, 1563-1573.\n\u003c/p\u003e\u003cp\u003eSeed, J., Carney, E.W., Corley, R.A., Crofton, K.M., DeSesso, J.M., Foster, P.M., Kavlock, R., Kimmel, G., Klaunig, J., Meek, M.E., Preston, R.J., Slikker, W. Jr., Tabacova, S., Williams, G.M., Wiltse, J., Zoeller, R.T., Fenner-Crisp, P., Patton, D.E. (2005). Overview: using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit. Rev. Toxicol. 35, 664-672.\n\u003c/p\u003e\u003cp\u003eSekine, S., Yano K.,, Saeki, J., Hashimoto, N., Fuwa, T., Horie, T. (2010). Oxidative stress is a triggering factor for LPS-induced Mrp2 internalization in the cryopreserved rat and human liver slices. Biochem. Biophys. Res. Commun. 399, 279-285.\n\u003c/p\u003e\u003cp\u003eSeok, J., Warren, H.S., Cuenca, A.G., Mindrinos, M.N., Baker, H.V., Xu, W., Richards, D.R., McDonald-Smith, G.P., Gao, H., Hennessy, L., Finnerty, C.C., López, C.M., Honari, S., Moore, E.E., Minei, J.P., Cuschieri, J., Bankey, P.E., Johnson, J.L., Sperry, J., Nathens, A.B., Billiar, T.R., West, M.A., Jeschke, M.G., Klein, M.B., Gamelli, R.L., Gibran, N.S., Brownstein, B.H., Miller-Graziano, C., Calvano, S.E., Mason, P.H., Cobb, J.P., Rahme, L.G., Lowry, S.F., Maier, R.V., Moldawer, L.L., Herndon, D.N., Davis, R.W., Xiao, W., Tompkin, R.G. (2013). Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc. Natl. Acad. Sci. USA 110, 3507-3512.\n\u003c/p\u003e\u003cp\u003eSokol, R.J., Dahl, R., Devereaux, M.W., Yerushalmi, B., Kobak, G.E., Gumpricht, E. (2005). Human hepatic mitochondria generate reactive oxygen species and undergo the permeability transition in response to hydrophobic bile acids. J. Pediatr. Gastroenterol. Nutr. 41, 235-243.\n\u003c/p\u003e\u003cp\u003eTrauner, M., Arrese, M., Lee, H., Boyer, J.L., Karpen, S.J. (1998a). Endotoxin downregulates rat hepatic ntcp gene expression via decreased activity of critical transcription factors. J. Clin. Invest. 101, 2092-2100.\n\u003c/p\u003e\u003cp\u003eTrauner, M., Meier, P.J., Boyer, J.L. (1998b). Molecular pathogenesis of cholestasis. N. Engl. J. Med. 339, 1217-1227.\n\u003c/p\u003e\u003cp\u003eUS EPA. (2005). Guidelines for carcinogen risk assessment. Washington DC.\n\u003c/p\u003e\u003cp\u003eVinken, M., Pauwels, M., Ates, G., Vivier, M., Vanhaecke, T., Rogiers, V. (2012). Screening of repeated dose toxicity data present in SCC(NF)P/SCCS safety evaluations of cosmetic ingredients. Arch. Toxicol. 86, 405-412.\n\u003c/p\u003e\u003cp\u003eWagner, M., Zollner, G., Trauner, M. (2009). New molecular insights into the mechanisms of cholestasis. J. Hepatol. 51, 565-580.\n\u003c/p\u003e\u003cp\u003eWoolbright, B.L., Jaeschke, H. (2012). Novel insight into mechanisms of cholestatic liver injury. World. J. Gastroenterol. 18, 4985-4993.\n\u003c/p\u003e\u003cp\u003eYasumiba, S., Tazuma, S., Ochi, H., Chayama, K., Kajiyama, G. (2001). Cyclosporin A reduces canalicular membrane fluidity and regulates transporter function in rats. Biochem. J. 354, 591-596.\n\u003c/p\u003e\u003cp\u003eZhang, Y., Hong, J.Y., Rockwell, C.E., Copple, B.L., Jaeschke, H., Klaassen, C.D. (2012). Effect of bile duct ligation on bile acid composition in mouse serum and liver. Liver Int. 32, 58-69.\n\u003c/p\u003e\u003cp\u003eZollner, G., Wagner, M., Moustafa, T., Fickert, P., Silbert, D., Gumhold, J., Fuchsbichler, A., Halilbasic, E., Denk, H., Marschall, H.U., Trauner, M. (2006). Coordinated induction of bile acid detoxification and alternative elimination in mice: role of FXR-regulated organic solute transporter-alpha/beta in the adaptive response to bile acids. J. Physiol. Gastrointest. Liver Physiol. 290, G923-G932.\n\u003c/p\u003e\u003cp\u003eZollner, G., Trauner, M. (2006). Molecular mechanisms of cholestasis. Wien. Med. Wochenschr. 156, 380-385.\n\u003c/p\u003e\u003cp\u003eZollner, G., Trauner, M. (2008). Mechanisms of cholestasis. Clin. Liver Dis. 12, 1-26.\n\u003c/p\u003e\n\u003ch2\u003e\u003cspan class=\"mw-headline\" id=\"Confidence_in_the_AOP\"\u003eConfidence in the AOP\u003c/span\u003e\u003c/h2\u003e\n\u003cp\u003e\u003cspan style=\"color:red; margin:30px; font-weigth:900; font-size: 200%\"\u003eInformation from this section should be moved to the Key Event Relationship pages!\u003cbr /\u003e\u003cbr /\u003e\u003c/span\u003e\n1. How well characterized is the AOP?\nDrug-induced cholestasis is a well understood AO that is causally and dose-dependently linked to BSEP inhibition, being the predominant MIE (Dawson et al., 2011; Kis et al., 2012; Morgan et al., 2010). Furthermore, the critical role of the key events, namely the accumulation of bile, the induction of inflammation and oxidative stress and the activation of specific nuclear receptors, as well as the different intermediate steps in the AOP, is supported by a wealth of experimental data. Thus, despite a number of limitations in scientific evidence, as will be discussed further, the established general structure and components of the AOP can be considered as being well-characterized.\n\u003c/p\u003e\u003cp\u003e2. How well are the initiating and other key events causally linked to the outcome?\nIt has been demonstrated on numerous occasions that BSEP inhibition is causally linked to the induction of cholestasis in a dose-dependent way, both in experimental animals and in humans (Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). BSEP inhibition directly leads to bile accumulation, which subsequently activates an inflammatory reaction as well as the occurrence of oxidative stress (Gujral et al., 2003 and 2004; Jaeschke and Hasegawa, 2006; Woolbright and Jaeschke, 2012). The resulting cell death and associated bile acid-induced membrane damage of hepatocytes and cholangiocytes underlie the increased serum concentrations of ALT, AST, ALP, GGT and 5’-NT, being a prominent clinical hallmark of cholestasis (Hofmann, 2009; Kuntz and Kuntz, 2008; Padda et al., 2011). To compensate for the cholestatic insults, an adaptive response is induced, which is initiated by nuclear receptor activation and that is targeted towards the elimination of bile from the organism (Boyer, 2009; Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). Consequently, elevated concentrations of bilirubin in serum and urine are observed. The former causes jaundice, while the increased presence of bile acids in serum is thought to induce pruritus (Hofmann, 2009; Kuntz and Kuntz, 2008; Padda et al., 2011; Zollner and Trauner, 2008). Thus, there is a direct, causal and, at least in some cases, quantitative association between the MIE, the key events and the AO in the established AOP.\n\u003c/p\u003e\u003cp\u003e3. What are the limitations in the evidence in support of the AOP?\nThere are a number of limitations in the scientific evidence in support of the AOP in relation to temporal concordance of the intermediate steps and key events, the consistency of the available experimental data, alternative mechanisms involved, interspecies and intraspecies differences, uncertainties, inconsistencies and data gaps. These shortcomings are addressed in previous sections.\n\u003c/p\u003e\u003cp\u003e4. Is the AOP specific to certain tissues, life stages or age classes?\nAlthough the entire process of drug-induced cholestasis mainly takes place in the liver, more specifically in hepatocytes, the adaptive changes to this insult also occur in other tissues, including the kidney and the intestine. In this context, expression of MRP2 is induced in renal tubular cells in experimental models of cholestasis (Lee et al., 2001). Alterations in transporter protein expression during cholestasis also occur in other tissues, such as the ileum (Mennone et al., 2010). Like in the liver, the overall goal of these alterations is to increase elimination of bile salts via the urine and feces (Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). Regarding age-specificity, no significant quantitative differences in BSEP expression between fetal and human liver have been detected (Chen et al., 2005). Hepatocellular accumulation of bile acids causes giant cell hepatitis and progressive liver damage in children (Oude Elferink et al., 2006), which may burgeon into hepatocellular carcinoma (Bernstein et al., 2005; Knisely et al., 2006). On the other hand, it is well established that age over 50 years poses an increased risk to develop drug-induced hepatic damage (Pauli-Magnus and Meier, 2006) and that DILI in elder people is of cholestatic rather than of hepatocelluar nature (Lucena et al., 2009). In general, women are more susceptible for developing DILI than men (Pauli-Magnus and Meier, 2006), yet no gender differences exist in liver BSEP expression in humans (Cheng et al., 2007). In contrast, male rats are more prone to troglitazone-induced cholestasis than female rats because of higher rates of troglitazone sulphate formation (Funk et al., 2001; Kostrubsky et al., 2001). At the population level, genetic variability in the BSEP gene, leading to its decreased expression, may predispose different ethnic populations to drug-induced cholestasis (Lang et al., 2006 and 2007; Meier et al., 2006).\n\u003c/p\u003e\u003cp\u003e5. Are the initiating and key events expected to be conserved across taxa?\nStandard animal studies conducted during drug development, using mainly rodents, usually pick up about half of all human hepatotoxic compounds because of interspecies differences (Blomme et al., 2009; Ozer et al., 2008). In the case of BSEP inhibition, however, interspecies differences are mostly of quantitative nature. This could be due to the fact that there is a high amino acid similarity between human BSEP and its rodent counterparts, namely 80% in mouse and 82% in rat (Green et al., 2000; Lecureur et al., 2000). Accordingly, while IC50 values for BSEP inhibition differ only minimally between human and mouse for troglitazone, they differ by almost an order of magnitude for glibenclamide (Kis et al., 2012). Other reasons for the absence of hepatotoxicity induced by human-relevant cholestatic drugs in rats include higher rates of basolateral bile salt efflux, which could represent an additional protective mechanism against cholestasis (Jemnitz et al., 2010). In addition to BSEP inhibition, the key events of the proposed AOP are expected to be generally well conserved among taxa. Nevertheless, a recent report showed that considerable differences exist in inflammatory responses between human and mouse (Seok et al., 2013). Despite the occurrence of interspecies differences in their expression or ligand binding, such as shown for PXR (Krasowski et al., 2005), activation of nuclear receptors is a critical event in different animal models of cholestasis (Wagner et al., 2009; Zollner and Trauner, 2006 and 2008). It remains to be established whether data included in the AOP can be extrapolated from animals to humans and vice versa.\n\u003c/p\u003e","overall_assessment":"\u003cp\u003e1. Concordance of dose-response relationships: \nMorgan and colleagues investigated the potential of more than 200 benchmark drugs to inhibit BSEP. As much as 16% of the tested drugs displayed high potency of BSEP inhibition (IC50 ≤ 25 µM), the majority of which are associated with liver liabilities in humans (Morgan et al., 2010). Likewise, 17 of 85 pharmaceuticals tested by Dawson and coworkers inhibited BSEP (IC50 ≤ 100 µM), all which are known to cause DILI (Dawson et al., 2011). Furthermore, several of the BSEP-inhibiting drugs cause cholestastic liver injury in a dose-dependent way, such as is the case for troglitazone and bosentan in rats (Funk et al., 2001) and humans (Fattinger et al., 2001), respectively. Thus, there is a clear relationship between the IC50 of BSEP inhibition and the occurrence of (cholestatic) DILI.\n\u003c/p\u003e\u003cp\u003e2. Temporal concordance among the key events and adverse effect: \nInhibition of BSEP activity and the resulting accumulation of bile acids primarily triggers a direct cellular response, which is associated with deteriorative processes, such as inflammation, oxidative stress and cell death. It also causes a secondary and rather indirect cellular response, which is adaptive in nature. Indeed, a well-orchestrated network of mechanisms is activated, all of which are targeted towards the elimination of bile from the liver (Boyer, 2009; Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). The temporal concordance between these cellular responses is not clear. It is, however, conceivable to assume that the adaptive response becomes manifested at a somewhat later stage when compared to the primary events, especially since this secondary response highly depends on transcriptional regulation. Nonetheless, it is clear that the graphical linear representation of cholestatic DILI resulting from BSEP inhibition as a sequence of events is an oversimplification of a probably very complex network of entangled consecutive and parallel reactions.\n\u003c/p\u003e\u003cp\u003e3. Strength, consistency, and specificity of association of adverse effect and initiating event: \nBSEP is considered as the major apical transporter protein that pumps bile salts from hepatocytes into bile canaliculi. As a part of this pivotal task, BSEP has a very narrow substrate specificity with only a few known non-bile substrates (Dawson et al., 2011; Kis et al., 2012; Morgan et al., 2010). Defects in BSEP expression or function therefore can be anticipated to have drastic consequences with respect to bile homeostasis. Indeed, a plethora of studies has demonstrated that BSEP inhibition or impairment is causally linked to the induction of cholestasis in a dose-dependent way, both in experimental animals and in humans (Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). Thus, there is a well-established, direct, specific and quantitative association between BSEP inhibition and the onset of cholestatic DILI.\n\u003c/p\u003e\u003cp\u003e4. Biological plausibility, coherence, and consistency of the experimental evidence: \nIn essence, BSEP inhibition (i.e. the MIE) activates a number of mechanisms that drive a deteriorative cellular response, which underlies directly caused cholestatic injury, as well as an adaptive cellular response, which is aimed at counteracting cholestatic insults. Both these responses contribute to the clinical manifestation of cholestasis (i.e. the AO). Serum concentrations of ALT, AST, ALP, GGT and 5’-NT indeed increase because of bile acid-induced membrane damage of hepatocytes and cholangiocytes (Hofmann, 2009; Kuntz and Kuntz, 2008; Padda et al., 2011). At the same time, elevated concentrations of bilirubin in serum and urine are observed, reflecting the compensatory response of the organism to counteract bile acid accumulation. Hyperbilirubinemia causes jaundice, while the increased presence of bile acids in serum is thought to induce pruritus (Hofmann, 2009; Kuntz and Kuntz, 2008; Padda et al., 2011; Zollner and Trauner, 2008).\n\u003c/p\u003e\u003cp\u003e5. Alternative mechanisms that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP: \nAlthough predominant, BSEP inhibition is not the sole MIE in cholestatic DILI, as depicted in the established AOP. In this regard, cyclosporine A not only inhibits BSEP (Dawnson et al., 2011; Kis et al., 2012; Morgan et al., 2010), but also induces cholestasis by inhibition of intrahepatic vesicle transport (Roman et al., 1990) and by affecting canalicular membrane fluidity (Yasumiba et al., 2001). Several of these events, in particular the cytoskeletal changes, might be considered as secondary and non-specific phenomena (Trauner et al., 1998b). Nevertheless, separate AOPs could be drafted for each of these alternative mechanisms in cholestatic DILI.\n\u003c/p\u003e\u003cp\u003e6. Uncertainties, inconsistencies and data gaps: \nAlthough a clear causal and dose-dependent relationship has been established between BSEP inhibition and the clinical onset of cholestasis (Zollner and Trauner, 2006 and 2008; Wagner et al., 2009), several mechanisms of the intermediate steps and key events as well as their linkage are not fully understood. A prominent discussion in this respect relates to the nature of the cell death mode, namely apoptosis or necrosis, associated with cholestasis (Woolbright and Jaeschke, 2012). High concentrations of hydrophobic bile acids induce apoptotic cell death in cultures of primary hepatocytes (Gores et al., 1998; Botla et al., 1995; Schoemaker et al., 2004), yet such concentrations are not achieved in vivo (Zhang et al., 2012). It has therefore been suggested that the main mechanism of cell death in cholestasis in vivo is necrosis (Woolbright and Jaeschke, 2012). In fact, this seems to be a general consideration of the AOP, as several other constituting data also have been derived from in vitro experimentation and need to be substantiated in vivo. On the other hand, a number of data are still lacking, including the full identification of FXR, PXR and CAR target genes, which may additionally contribute to the adaptive response to BSEP inhibition. Furthermore, ongoing research regarding the regulation of these nuclear receptors in cholestatic DILI might add complexity to the AOP. It is known that they act, at least in part, by recruiting co-activators and co-repressors (Gollamudi et al., 2008; Wagner et al., 2009). Moreover, compelling evidence suggests that nuclear receptors are regulated epigenetically, which might necessitate inclusion in the AOP (Elloranta and Kullak-Ublick, 2005; Wagner et al., 2009). Additional uncertainties, inconsistencies and data gaps associated with the established AOP relate to the temporal concordance of the intermediate steps and key events, the consistency of the available experimental data, alternative mechanisms involved, interspecies and intraspecies differences.\n\u003c/p\u003e","background":"","user_defined_mie":"41: Inhibition, Bile Salt Export Pump (ABCB11)","user_defined_ao":"357: Cholestasis, Pathology","oecd_project":"1.19","oecd_status_id":4,"graphical_representation_image_uid":"2016/12/02/6uxaasyru1_Cholestatic_Liver_Injury_induced_by_Inhibition_of_the_Bile_Salt_Export_Pump__ABCB11_.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2025-04-03T16:42:59.000-04:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":22,"handbook_id":1,"project_129":false},{"id":28,"title":"Cyclooxygenase inhibition leading reproductive failure","short_name":"Cyclooxygenase inhibition leading reproductive failure","corresponding_author_id":313,"abstract":"","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2026-01-11T16:56:06.000-05:00","status_id":6,"authors":"\u003cp\u003eDan Villeneuve, US EPA Mid-Continent Ecology Division (villeneuve.dan@epa.gov)\u003c/p\u003e\r\n","applicability_of_the_aop":"","key_event_essentiality":"","weight_of_evidence_summary":"","quantitative_considerations":"","optional_considerations":"","references":"","overall_assessment":"\u003cp\u003e\u003cem\u003eConsider the following criteria (may include references to KE Relationship pages): 1. concordance of dose-response relationships; 2. temporal concordance among the key events and adverse effect; 3. strength, consistency, and specificity of association of adverse effect and initiating event; 4. biological plausibility, coherence, and consistency of the experimental evidence; 5. alternative mechanisms that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP; 6. uncertainties, inconsistencies and data gaps. \u003c/em\u003e\u003c/p\u003e\r\n","background":"","user_defined_mie":"79: Inhibition, Cyclooxygenase activity","user_defined_ao":"253: N/A, Reproductive failure","oecd_project":null,"oecd_status_id":null,"graphical_representation_image_uid":null,"saaop_status_id":3,"legacy":true,"overall_assessment_file_uid":null,"changed_at":null,"development_strategy":null,"known_modulating_factors":null,"assigned_license_id":23,"handbook_id":1,"project_129":true},{"id":29,"title":"Estrogen receptor agonism leading to reproductive dysfunction","short_name":"Estrogen receptor agonism leading to reproductive dysfunction","corresponding_author_id":null,"abstract":"\u003cp\u003eThis AOP describes the linkages between agonism of the estrogen receptor (ER) and population relevant impacts on reproductive function in a range of oviparous vertebrates including amphibia, birds and fish. The information in this AOP for ER agonism does not apply to mammalian species and also not to invertebrates.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cbr /\u003e\r\nAmphibians are sensitive to ER agonists during the transformation from larval tadpole to juvenile frog as these include critical periods of metamorphic development and sex differentiation that may be particularly sensitive to endocrine disruption. Larvae exposed to ER agonists during mid-metamorphosis show developmental effects, a subsequent strong female-biased sex ratio which suggests that transient early life-stage exposure to ER agonists can produce effects on the reproductive organs that persist into the beginning of adult life-stages. Birds are also known to be vulnerable to ER agonists causing disruption of estrogen-regulated functions such as sexual differentiation and sexual behaviour. Model species such as the Japanese quail have been widely used as a model for studying various long-term effects after embryonic exposure to ER agonists. In terms of teleost fish, exposure to ER agonists leads to a suite of adverse outcomes depending upon whether exposures occur during or beyond the larval, juvenile and adult life-stages. For example, aquatic exposure to potent ER agonists during the larval and juvenile life-stages may leads to gonadal and renal pathology and skewed-sex ratios in adult fish (potentially 100% females). Larval, juvenile and adult male fish exposed to the same ER agonists display abnormal plasma or whole body levels of vitellogenin (VTG). Cumulative fecundity in adult populations is also adversely affected by ER agonists and this is an important endpoint in the OECD Test Guideline 229 Fish Short Term Reproduction Assay. In summary, this AOP has utility in supporting the application of test methods for detecting ER agonists, or in silico predictions of the ability of chemicals to act as ER agonists and cause impaired sexual development and reproductive dysfunction.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2026-01-11T16:56:06.000-05:00","status_id":6,"authors":"\u003cul\u003e\r\n\t\u003cli\u003eProfessor Tom Hutchinson, School of Biological Sciences, Plymouth, UK [tom.hutchinson{at}plymouth.ac.uk]\u003c/li\u003e\r\n\t\u003cli\u003eDan Villeneuve, US EPA Mid-Continent Ecology Division, Duluth, MN. [villeneuve.dan{at}epa.gov]\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","applicability_of_the_aop":"\u003cp\u003e\u003ca href=\"#Life_Stage_Applicability\"\u003eLife Stage Applicability\u003c/a\u003e, \u003ca href=\"#Taxonomic_Applicability\"\u003e Taxonomic Applicability\u003c/a\u003e, \u003ca href=\"#Sex_Applicability\"\u003e Sex Applicability\u003c/a\u003e\u003cbr /\u003e\r\nIn terms of the taxonomic domains of applicability, exposure to ER agonists is capable of disrupting sexual development and causing reproductive dysfunction in oviparous species suchas amphibians, birds and fish (see examples of peer-revised literature cited below).\u003c/p\u003e\r\n","key_event_essentiality":"","weight_of_evidence_summary":"","quantitative_considerations":"","optional_considerations":"","references":"\u003cp\u003e\u003cbr /\u003e\r\nDang, Z., Traas, T., Vermeire, T. (2011) Evaluation of the fish short term reproduction assay for detecting endocrine disrupters. Chemosphere 85: 1592-1603\u003c/p\u003e\r\n\r\n\u003cp\u003eHalldin, K., Axelsson, J., Brunstr\u0026ouml;m, B., (2005) Effects of endocrine modulators on sexual differentiation and reproductive function in male Japanese quail. Brain Research Bulletin 65: 211-218\u003c/p\u003e\r\n\r\n\u003cp\u003eHogan, N.S., Duarte, P., Wade, M.G., Lean, D.R.S., Trudeau, V.L. (2008) Estrogenic exposure affects metamorphosis and alters sex ratios in the northern leopard frog (Rana pipiens): Identifying critically vulnerable periods of development. General and Comparative Endocrinology 156: 515-523\u003c/p\u003e\r\n\r\n\u003cp\u003eHutchinson T.H. (2002) Impacts of endocrine disrupters on fish development: opportunities for adapting OECD Test Guideline 210. Environmental Sciences 9: 439-450\u003c/p\u003e\r\n\r\n\u003cp\u003eL\u0026auml;nge R., Hutchinson T.H., Croudace C.P., Siegmund F., Schweinfurth H., Hampe P., Panter G.H., Sumpter J.P. (2001) Effects of the synthetic oestrogen 17-ethinylestradiol over the life-cycle of the fathead minnow. Environmental Toxicology and Chemistry 20: 1216\u0026ndash;1227\u003c/p\u003e\r\n\r\n\u003cp\u003eLeino, R.L., Jensen,K.M., Ankley, G.T. (2005) Gonadal histology and characteristic histopathology associated with endocrine disruption in the adult fathead minnow (Pimephales promelas). Environmental Toxicology and Pharmacology 19: 85-98\u003c/p\u003e\r\n\r\n\u003cp\u003eOttinger, M.N., Carro, T., Bohannon, M., Baltos,L., Marcell, A.M., McKernan, M., Dean, K.M., Lavoie, E., Abdelnabi, M. (2013) Assessing effects of environmental chemicals on neuroendocrine systems: Potential mechanisms and functional outcomes. General and Comparative Endocrinology 190: 194-202\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003eIn terms of the criteria associated with Key Events in this AOP, the following observations have been made as shown in parentheses []:\u003c/p\u003e\r\n\r\n\u003cp\u003e1. concordance of dose-response relationships?; [There is strong dose-response relationship concordance over a wide range of experimental studies using ER agonists in well-defined animals models, including amphibians, birds and fish];\u003c/p\u003e\r\n\r\n\u003cp\u003e2. temporal concordance among the key events and adverse effect?; [There is strong temporal concordance from partial and full life-cycle studies using ER agonists in well-defined animals models];\u003c/p\u003e\r\n\r\n\u003cp\u003e3. strength, consistency, and specificity of association of adverse effect and initiating event?; [In fish, there is a strong and consistent association between ER agonist exposure, disruption of sexual development and reproductive dysfunction. The same is true for amphibians and birds although the published studies are less numerous.];\u003c/p\u003e\r\n\r\n\u003cp\u003e4. biological plausibility, coherence, and consistency of the experimental evidence?; [For the oviparous species frequently studied to date, there is a high level of biological plausibility, coherence, and consistency across the published experimental evidence];\u003c/p\u003e\r\n\r\n\u003cp\u003e5. alternative mechanisms that logically present themselves and the extent to which they may distract from the postulated AOP?; [Other mechanisms of relevance to estrogen-mediated sexual development include the disruption of the steroidogenic pathways (eg see the AOP for aromatase inhibition in fish) and this alterative AOP should be considered alongside ER agonism in the context of elevated plasma VTG levels, disrupted sexual development of reproductive dysfunction. The possibility of other AOPs arisign should be kept in mind through critical analysis of the updated pree-reviewed literature];\u003c/p\u003e\r\n\r\n\u003cp\u003e6. uncertainties, inconsistencies and data gaps?; [An important aspect of uncertainty is quantifying the degree to which disrupted sexual development leads to a population-relevant impact via reproductive dysfunction. Experimental and validated population modelling is a key need to address this data gap and uncertainty. In the author\u0026#39;s view, there are no major scientific inconsistencies with regard to the ER agonism AOP and associated Key Events].\u003c/p\u003e\r\n","background":"","user_defined_mie":"111: Agonism, Estrogen receptor","user_defined_ao":"360: Decrease, Population trajectory and 363: Altered, Reproductive behaviour and 339: Altered, Larval development and 364:  Impaired development of, Reproductive organs","oecd_project":null,"oecd_status_id":null,"graphical_representation_image_uid":null,"saaop_status_id":3,"legacy":true,"overall_assessment_file_uid":null,"changed_at":null,"development_strategy":null,"known_modulating_factors":null,"assigned_license_id":24,"handbook_id":1,"project_129":true},{"id":30,"title":"Estrogen receptor antagonism leading to reproductive dysfunction","short_name":"Estrogen receptor antagonism leading to reproductive dysfunction","corresponding_author_id":313,"abstract":"\u003cp\u003eThis adverse outcome pathway details the linkage between antagonism of estrogen receptor in females and the adverse effect of reduced cumulative fecundity in repeat-spawning fish species. Cumulative fecundity is the most apical endpoint considered in the OECD 229 Fish Short Term Reproduction Assay. The OECD 229 assay serves as screening assay for endocrine disruption and associated reproductive impairment (OECD 2012a). Cumulative fecundity is one of several variables known to be of demographic significance in forecasting fish population trends. Therefore, this AOP has utility in supporting the application of measures of ER antagonism, or in silico predictions of the ability to antagonize ER as a means to identify chemicals with known potential to adversely affect fish populations.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":1,"authors":"\u003cp\u003eDaniel L. Villeneuve, US EPA Mid-Continent Ecology Division (villeneuve.dan@epa.gov)\u003c/p\u003e\r\n","applicability_of_the_aop":"\u003cp\u003e\u003cstrong\u003eLife Stage\u003c/strong\u003e: This AOP applies to sexually mature animals. \u003cstrong\u003eSex\u003c/strong\u003e: This AOP applies to females. \u003cstrong\u003eTaxonomic Applicability\u003c/strong\u003e: Based on the taxonomic applicability of the component key events, this AOP could potentially apply to most oviparous chordates.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eDomain(s) of Applicability\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eSex\u003c/strong\u003e: The AOP applies to females only\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eLife stages\u003c/strong\u003e: The relevant life stages for this AOP are reproductively mature adults. This AOP does not apply to adult stages that lack a sexually mature ovary, for example as a result of seasonal or environmentally-induced gonadal senescence (i.e., through control of temperature, photo-period, etc. in a laboratory setting).\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eTaxonomic\u003c/strong\u003e: At present, the assumed taxonomic applicability domain of this AOP is class Osteichthyes. In all likelihood, the AOP will also prove applicable to all classes of fish (e.g., Agnatha and Chondrithyes as well). Additionally, all the key events described should be conserved among all oviparous vertebrates, suggesting that the AOP may also have relevance for amphibians, reptiles, and birds. However, species-specific differences in reproductive strategies/life histories, ADME (adsorption, distribution, metabolism, and elimination), compensatory reproductive endocrine responses may influence the outcomes, particularly from a quantitative standpoint.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","key_event_essentiality":"","weight_of_evidence_summary":"\u003cp\u003eThe weight of evidence for each of the KERs comprising the AOP are ranked moderate to strong. Biological plausibility at the molecular and cellular level of the early key events is very strong. Some uncertainties regarding the mechanistic details of the connection between reduced vtg availability and uptake limit the strength of evidence to some degree. However, there are considerable evidence to support the idea that ER antagonism can ultimately lead to reproductive failure. Overall weight of evidence is moderate.\u003c/p\u003e\r\n","quantitative_considerations":"\u003cp\u003eA quantitative relationship between ER antagonism (the MIE) and reductions in vitellogenin transcription and translation have not been well established. However, a correlative relationship between plasma vitellogenin concentrations and cumulative fecundity has been reported (Miller et al. 2007) and applied for quantitative modeling (Ankley et al.\u003c/p\u003e\r\n","optional_considerations":"","references":"\u003cul\u003e\r\n\t\u003cli\u003eOECD. 2012a. Test no. 229: Fish short term reproduction assay. Paris, France:Organization for Economic Cooperation and Development.\u003c/li\u003e\r\n\t\u003cli\u003eWester P, van den Brandhof E, Vos J, van der Ven L. 2003. Identification of endocrine disruptive effects in the aquatic environment - a partial life cycle assay in zebrafish. (RIVM Report). Bilthoven, the Netherlands:Joint Dutch Environment Ministry\u003c/li\u003e\r\n\t\u003cli\u003eSun L, Zha J, Spear PA, Wang Z. 2007b. Tamoxifen effects on the early life stages and reproduction of japanese medaka (oryzias latipes). Environmental toxicology and pharmacology 24:23-29.\u003c/li\u003e\r\n\t\u003cli\u003eSun L, Zha J, Wang Z. 2009. Effects of binary mixtures of estrogen and antiestrogens on japanese medaka (oryzias latipes). Aquatic toxicology 93:83-89.\u003c/li\u003e\r\n\t\u003cli\u003eWilliams TD, Caunter JE, Lillicrap AD, Hutchinson TH, Gillings EG, Duffell S. 2007. Evaluation of the reproductive effects of tamoxifen citrate in partial and full life-cycle studies using fathead minnows (pimephales promelas). Environmental toxicology and chemistry / SETAC 26:695-707.\u003c/li\u003e\r\n\t\u003cli\u003evan der Ven LT, van den Brandhof EJ, Vos JH, Wester PW. 2007. Effects of the estrogen agonist 17beta-estradiol and antagonist tamoxifen in a partial life-cycle assay with zebrafish (danio rerio). Environmental toxicology and chemistry / SETAC 26:92-99.\u003c/li\u003e\r\n\t\u003cli\u003eChikae M, Ikeda R, Hasan Q, Morita Y, Tamiya E. 2004. Effects of tamoxifen, 17alpha-ethynylestradiol, flutamide, and methyltestosterone on plasma vitellogenin levels of male and female japanese medaka (oryzias latipes). Environmental toxicology and pharmacology 17:29-33.\u003c/li\u003e\r\n\t\u003cli\u003eLeanos-Castaneda O, Van Der Kraak G. 2007. Functional characterization of estrogen receptor subtypes, eralpha and erbeta, mediating vitellogenin production in the liver of rainbow trout. Toxicology and applied pharmacology 224:116-125.\u003c/li\u003e\r\n\t\u003cli\u003eGriffin LB, January KE, Ho KW, Cotter KA, Callard GV. 2013. Morpholino mediated knockdown of eralpha, erbetaa and erbetab mrnas in zebrafish (danio rerio) embryos reveals differential regulation of estrogen-inducible genes. Endocrinology.\u003c/li\u003e\r\n\t\u003cli\u003eDavis LK, Katsu Y, Iguchi T, Lerner DT, Hirano T, Grau EG. 2010. Transcriptional activity and biological effects of mammalian estrogen receptor ligands on three hepatic estrogen receptors in mozambique tilapia. The Journal of steroid biochemistry and molecular biology 122:272-278.\u003c/li\u003e\r\n\t\u003cli\u003eNagler JJ, Cavileer TD, Verducci JS, Schultz IR, Hook SE, Hayton WL. 2012. Estrogen receptor mrna expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle. General and comparative endocrinology 178:556-561.\u003c/li\u003e\r\n\t\u003cli\u003eNelson ER, Habibi HR. 2010. Functional significance of nuclear estrogen receptor subtypes in the liver of goldfish. Endocrinology 151:1668-1676.\u003c/li\u003e\r\n\t\u003cli\u003eMiller DH, Jensen KM, Villeneuve DL, Kahl MD, Makynen EA, Durhan EJ, Ankley GT. 2007. Linkage of biochemical responses to population-level effects: a case study with vitellogenin in the fathead minnow (\u003cem\u003ePimephales promelas\u003c/em\u003e). Environ. Toxicol. Chem. 26: 521-527.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n","overall_assessment":"\u003cul\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eOverall Assessment of the AOP\u003c/strong\u003e\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eConcordance of dose-response relationships\u003c/strong\u003e:\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eIn a 42 d static renewal exposure to tamoxifen, significant, concentration dependent reduction in the number of clutches and cumulative fecundity were observed for zebrafish (Wester et al. 2003).\u003c/li\u003e\r\n\t\u003cli\u003eA concentration-dependent reduction in circulating vitellogenin concentrations was detected in female medaka exposed to tamoxifen for 21 d (Sun et al. 2007b). Vitellogenin reductions occurred at a lower concentration (i.e., \u0026ge; 25 \u0026mu;g tamoxifen/L) than reductions in fecundity (i.e., 625 \u0026mu;g tamoxifen/L).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eTemporal concordance among the key events and adverse effect\u003c/strong\u003e: To date, there are no time-course studies that allow for robust evaluation of the temporal concordance of the entire AOP. However, the temporal concordance of some of the key event relationships has been established. Specifically, reductions in transcription of vitellogenin mRNAs have been shown to precede changes in circulating vitellogenin concentrations.\u003c/li\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eConsistency\u003c/strong\u003e:\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eIn zebrafish exposed to tamoxifen, reductions in the number of clutches and cumulative egg production were predicted to result in population reductions, although this was in conjunction with altered sex ratios as a concurrent effect in a partial life-cycle test (Wester et al. 2003).\u003c/li\u003e\r\n\t\u003cli\u003eIn medaka co-exposed to 17\u0026beta;-estradiol (E2; 200 ng/L) and 10, 50, or 250 \u0026mu;g tamoxifen/L, exposure to 250 \u0026mu;g tamoxifen significantly reduced fecundity compared to both controls and fish exposed to E2 alone (Sun et al. 2009).\u003c/li\u003e\r\n\t\u003cli\u003eFecundity was significantly reduced in medaka exposed to 625 \u0026mu;g tamoxifen/L (Sun et al. 2007b).\u003c/li\u003e\r\n\t\u003cli\u003eIncreases in atretic oocytes and oviducts filled with degenerated eggs were observed in female zebrafish exposed to tamoxifen (Wester et al. 2003). Reduced vitellogenin immuno staining was observed in tamoxifen-exposed zebrafish, based on blind semi-quantitative scoring (van der Ven et al. 2007; Wester et al. 2003). The results are therefore consistent with the AOP.\u003c/li\u003e\r\n\t\u003cli\u003eIn Japanese medaka co-exposed to E2 and tamoxifen for 21 d, both plasma vitellogenin and fecundity were reduced in a tamoxifen concentration-dependent manner (Sun et al. 2009). Although from a co-exposure, the results are broadly consistent with the AOP.\u003c/li\u003e\r\n\t\u003cli\u003eIn Japanese medaka exposed to tamoxifen for 21 d, plasma vitellogenin in females was reduced in a concentration-dependent manner and cumulative fecundity was reduced at the maximum concentration tested (Sun et al. 2007b). The results are consistent with the AOP.\u003c/li\u003e\r\n\t\u003cli\u003eDietary exposure to tamoxifen was also shown to reduce circulating vitellogenin concentrations in female medaka (Chikae et al. 2004). The results are consistent with the AOP.\u003c/li\u003e\r\n\t\u003cli\u003eIn tilapia co-injected with E2 or o,p-DDT, tamoxifen inhibited the stimulatory effects of E2 and o,p-DDT on plasma vitellogenin (measured as alkaline labile phosphorous). Alkaline labile phosphorous was not reduced following injection with tamoxifen alone (Leanos-Castaneda et al. 2002). These results are neither entirely consistent nor inconsistent with the AOP.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003e\u003cstrong\u003eUncertainties, inconsistencies, and data gaps\u003c/strong\u003e:\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eIn a 42 d in vivo, flow through, exposures to tamoxifen citrate, no significant reductions in circulating vitellogenin or cumulative fecundity were detected (Williams et al. 2007). The results are therefore inconsistent with the AOP.\u003c/li\u003e\r\n\t\u003cli\u003eSome uncertainty remains regarding which ER subtype(s) regulates vitellogenin gene expression in the liver of fish. In general, the literature suggests a close interplay between several ER subtypes in the regulation of vitellogenesis. Consequently, at present, the AOP is generalized to impacts on all ER subtypes, even though it remains possible that impacts on a particular sub-type may drive the adverse response.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eGriffin et al. reported that morpholino knock-downs of either esr1 (ER\u0026alpha;) or esr2b (ER\u0026beta;b) prevented estradiol-mediated induction of vitellogenin expression in zebrafish (Griffin et al. 2013).\u003c/li\u003e\r\n\t\u003cli\u003eUsing selective agonists agonists and antagonists for ER\u0026alpha; and ER\u0026beta;, it was concluded that ER\u0026beta; was primarily responsible for inducing vitellogenin production in rainbow trout and that compounds exhibiting ER\u0026alpha; selectivity would not be detected using a vitellogenin bioassay (Leanos-Castaneda and Van Der Kraak 2007). However, a subsequent study conducted in tilapia concluded that agonistic and antagonistic characteristics of mammalian, isoform-specific ER agonists and antagonists, cannot be reliably extrapolated to piscine ERs (Davis et al. 2010).\u003c/li\u003e\r\n\t\u003cli\u003eExpression of both ER\u0026alpha;1 and ER\u0026beta;1 were strongly correlated with plasma vitellogenin concentrations over the reproductive cycle of rainbow trout (Nagler et al. 2012).\u003c/li\u003e\r\n\t\u003cli\u003eBased on RNA interference knock-down experiments Nelson and Habibi proposed a model in which all ER subtypes are involved in E2-mediated vitellogenesis, with ER\u0026beta; isoforms stimulating expression of both vitellogenin and ER\u0026alpha; gene expression, and ER\u0026alpha; helping to drive vitellogenesis, particularly as it becomes more abundant following sensitization (Nelson and Habibi 2010).\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cul\u003e\r\n\t\u003cli\u003eThere remains uncertainty as to whether there is a direct biological linkage, as opposed to correlation only, between impaired VTG uptake into oocytes and impaired spawning/reduced cumulative fecundity. Plausible biological connections have been hypothesized but have not yet been tested experimentally.\u003c/li\u003e\r\n\u003c/ul\u003e\r\n\r\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\r\n","background":"","user_defined_mie":"112: Antagonism, Estrogen receptor","user_defined_ao":"360: Decrease, Population trajectory","oecd_project":"1.12","oecd_status_id":3,"graphical_representation_image_uid":"2016/11/29/aa5Estrogen_receptor_antagonism_leading_to_reproductive_dysfunction.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":null,"development_strategy":null,"known_modulating_factors":null,"assigned_license_id":25,"handbook_id":1,"project_129":false},{"id":31,"title":"Oxidation of iron in hemoglobin leading to hematotoxicity","short_name":"Hemoglobin oxidation leading to hematotoxicity","corresponding_author_id":null,"abstract":"\u003cp\u003eStudies have shown that aniline, 2,4,6-trinitrotoluene (TNT) and 2,4-dinitrotoluene (2,4-DNT) are converted to the reactive metabolite and form free radicals leading to oxidization of heme Iron(II) in hemoglobin to Iron(III), a molecular initiating event. Damage then occurs to red blood cells (RBCs) and methemoglobinemia ensues which is characterized by reduced RBCs, hemoglobin concentration, and Heinz body formation (Ellis et al. 1985, Lee et al. 1976, 1978, Hazleton Laboratories 1977, 1982, Kozuka et al. 1978, 1979, Bolt et al. 2006). The adverse outcome due to such hematological effects is cyanosis with possible death if methemoglobin levels become severe. Hemoglobin adducts are also formed by these chemicals (Sabbioni et al. 2006). Sinusoidal congestion was noted in animals who were exposed to 2,4-DNT or 2,6-DNT (Deng et al. 2011) while hemosiderosis was reported in another study involving DNT (Lee et al. 1978) and in aniline studies. A compensatory response to possible anemic effects has been observed in animals including increased peripheral reticulocytes (Deng et al. 2011) and induction of genes associated with heme biosynthesis (CPOX and UROS) (Rawat et al. 2010). Oxidative stress is also induced upon this interaction with the RBC which may lead to DNA damage and cell death to not only the RBC but other cells such as hepatocytes (Deng et al. 2011). Glucose-6-phosphate dehydrogenase (G6pd) was found to be significantly down-regulated in animals treated with 2,4-DNT for 14 d which leads to decreased levels of NADPH, a coenzyme used to properly maintain glutathione levels and therefore protect cells, especially RBC, from oxidative damage (Wilbanks, et al., unpublished observations). In response to increased oxidative stress, protective mechanisms such as the Nrf2 mediated oxidative stress response may be induced (Deng et al. 2011). While this AOP specifically shows effects of 2,4-DNT and 2,6-DNT, the principal adverse pathways of oxidation of Fe(II) to Fe(III) leading to methemoglobinemia and its downsteam effects and oxidative stress formation leading to its downstream effects are shared with the more well characterized structurally similar compound group of N-hydroxyl anilines.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2024-07-24T15:26:29.000-04:00","status_id":3,"authors":"\u003cp\u003eMitchell Wilbanks, Kurt Gust, Youping Deng, Sharon Meyer, and Edward Perkins\u003c/p\u003e\r\n\r\n\u003cp\u003ePoint of Contact: Mitchell Wilbanks, Mitchell.S.Wilbanks@usace.army.mil\u003c/p\u003e\r\n","applicability_of_the_aop":"","key_event_essentiality":"","weight_of_evidence_summary":"","quantitative_considerations":"","optional_considerations":"","references":"\u003cp\u003eBolt HM, Degen GH, Dorn SB, Pl\u0026ouml;ttner S, Harth V (2006) Genotoxicity and potential carcinogenicity of 2,4,6-TNT trinitrotoluene: structural and toxicological considerations. Reviews on environmental health. Oct-Dec; 21(4):217-28.\u003c/p\u003e\r\n\r\n\u003cp\u003eDeng Y, Meyer SA, Guan X, Escalon BL, Ai J, et al. (2011) Analysis of Common and Specific Mechanisms of Liver Function Affected by Nitrotoluene Compounds. PLoS ONE. 6(2): e14662.\u003c/p\u003e\r\n\r\n\u003cp\u003eEllis HV, Hong CB, Lee CC, et al. 1985. Subchronic and chronic toxicity studies of 2,4-dinitrotoluene. Part I. Beagle dog. J Am Co11 Toxicol. 4:233-242.\u003c/p\u003e\r\n\r\n\u003cp\u003eJones, C.R., Liu, Y., Sepai, O., Yan, H., and Sabbioni, G. (2005). Hemoglobin adducts in workers exposed to nitrotoluenes. Carcinogenesis. 26(1):133-143.\u003c/p\u003e\r\n\r\n\u003cp\u003eKozuka H, Mori M., Katayama K, Matsuhashi T, Miyahara T, Mori Y, and Nagahara S. 1978. Studies on the metabolism and toxicity of dinitrotoluenes-Metabolism of dinitrotoluenes by Rhodotorula glutinis and rat liver homogenate. Eisei Kagaku, 24: 252-259.\u003c/p\u003e\r\n\r\n\u003cp\u003eKozuka H, Mori M, and Yoshifumi, N. 1979. Studies on the metabolism and toxicity of dinitrotoluenes: Toxicological study of 2,4-dinitrotoluene (2,4-DNT) in rats in long term feeding. The Journal of Toxicological Sciences. 4:221-228.\u003c/p\u003e\r\n\r\n\u003cp\u003eLa, D.K. and Froines, J.R. (1992). Comparison of DNA adduct formation between 2,4 and 2,6-dintirotoluene by 32P-postlabelling analysis. Archives of Toxicology. 66(9):633-640.\u003c/p\u003e\r\n\r\n\u003cp\u003eLee CC, Ellis HV, Kowalski JJ, et al. 1976. Mammalian toxicity of munitions compounds. Phase II: Effects of multiple doses. Part IIh 2,6-Dinitrotoluene. Progress report no. 4. Midwest Research Institute Project no. 3900-B. Contract no. DAMD-17-74-C-4073. From ASTDR.\u003c/p\u003e\r\n\r\n\u003cp\u003eLee CC, Ellis HV, Kowalski JJ, et al. 1978. Mammalian toxicity of munitions compounds. Phase II: Effects of multiple doses. Part Il: 2,4-Dinitrotoluene. Progress report No. 3. Midwest Research Institute, Kansas City, MO. Contract no. DAMD 17-74-C-4073. From ASTDR.\u003c/p\u003e\r\n\r\n\u003cp\u003eHazleton Laboratories. 1977. A thirty-day toxicology study in Fischer-344 rats given dinitrotoluene, technical grade. Full report. Submitted to Chemical Industry Institute of Toxicology, Research Triangle Park, NC.\u003c/p\u003e\r\n\r\n\u003cp\u003eHazleton Laboratories. 1982. 104-week chronic study in rats. Dinitrotoluene. Final report Volume I of II. Submitted to Chemical Industry Institute of Toxicology, Research Triangle Park, NC.\u003c/p\u003e\r\n\r\n\u003cp\u003eRawat A, Gust KA, Deng Y, Garcia-Reyero N, Quinn MJ Jr, Johnson MS, Indest KJ, Elasri MO, Perkins EJ. From raw materials to validated system: the construction of a genomic library and microarray to interpret systemic perturbations in Northern bobwhite. Physiol Genomics. 42: 219\u0026ndash;235, 2010.\u003c/p\u003e\r\n\r\n\u003cp\u003eSabbioni G, Jones CR, Sepai O, et al. 2006. Biomarkers of exposure, effect, and susceptibility in workers exposed to nitrotoluenes. Cancer Epidemiol Biomarkers. Prev 15(3):559-66.\u003c/p\u003e\r\n\r\n\u003cp\u003eWintz H, Yoo LJ, Loguinov A, Wu Y, Steevens JA, Holland RD, Beger RD, Perkins EJ, Hughes O, Vulpe CD. Gene expression profiles in fathead minnow exposed to 2,4-DNT: correlation with toxicity in mammals. Toxicol Sci. 94: 71\u0026ndash;82, 2006.\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003e\u003cem\u003eThis AOP is constructed using data from human, rat, mouse, avian, and fish based studies. \u003c/em\u003e\u003c/p\u003e\r\n","background":"","user_defined_mie":"213: N/A, Parent compound is converted to the reactive metabolite and forms free radicals leading to oxidation of heme iron(II) in hemoglobin to iron(III)","user_defined_ao":"321: N/A, Cyanosis occurs","oecd_project":"1.16","oecd_status_id":4,"graphical_representation_image_uid":"2016/11/29/55bHematotoxicity_due_to_nitroaromatics_and_N-hydroxyl_anilines.jpg","saaop_status_id":1,"legacy":true,"overall_assessment_file_uid":null,"changed_at":"2024-07-24T15:26:29.000-04:00","development_strategy":null,"known_modulating_factors":null,"assigned_license_id":26,"handbook_id":1,"project_129":false},{"id":32,"title":"Inhibition of iNOS, hepatotoxicity, and regenerative proliferation leading to liver tumors","short_name":"Inhibition of iNOS, hepatotoxicity, and regenerative proliferation leading to liver tumors","corresponding_author_id":null,"abstract":"\u003cp\u003eThiamethoxam is a neonicotinoid insecticide that has been extensively tested in animal models for short- and long-term toxicological effects. An increased incidence of liver tumors was seen in male and female Tif:MAGf mice when fed in the diet for 18 months at concentrations up to 2500 ppm. It is a mouse liver specific carcinogen and does not induce tumors at any other site in the mouse. There were no increases in cancer incidences either in the liver, or at any other site, in rats fed on diets containing up to 3000 ppm thiamethoxam for two years. Thiamethoxam was not genotoxic when evaluated in a battery of in vitro and in vivo assays.\u003c/p\u003e\r\n\r\n\u003cp\u003eThiamethoxam is metabolize to two key metabolites, CGA 322704 and CGA 330050. These metabolites can be further metabolize to CGA 265307. Basic toxicity studies on these metabolites give clues to the critical events involved in its mode of action resulting in hepatacarcinogenesis. These metabolites were given at doses to mimic systemic exposure that would result following a tumorigenic dose of Thiamethoxam. When administered directly in the rodent bioassay (rats and mice), the CGA 322704 and CGA 265307 metabolites did not result in any tumors or any other effect in the liver including altered serum cholesterol, liver toxicity, apoptosis, or increased cell proliferation. However, Metabolite CGA 265307 is very structurally similar with substrates and inhibitors of the nitric oxide synthases. Direct exposure to metabolite CGA 330050 did not result in tumors but did result in the same liver toxicity effects as for thiamethoxam. It is proposed that the metabolites CGA 330050 and CGA 265307 are involved in thiamethoxam\u0026rsquo;s hepatocarcinogensis.\u003c/p\u003e\r\n","created_at":"2016-11-29T18:41:16.000-05:00","updated_at":"2023-04-29T16:02:55.000-04:00","status_id":4,"authors":"\u003cp\u003eMichelle Embry, HESI\u003c/p\u003e\r\n","applicability_of_the_aop":"","key_event_essentiality":"","weight_of_evidence_summary":"","quantitative_considerations":"","optional_considerations":"","references":"\u003col\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-1\"\u003e\u0026uarr;\u003c/a\u003e Green, T., Toghill, A., Lee, R., Waechter, F., Weber, E., and Noakes, J. (2005a). Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 1: mode of action studies in the mouse. Toxicol. Sci. 86, 36\u0026ndash;47.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-2\"\u003e\u0026uarr;\u003c/a\u003e Green, T., Toghill, A., Lee, R., Waechter, F., Weber, E., and Noakes, J. (2005a). Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 1: mode of action studies in the mouse. Toxicol. Sci. 86, 36\u0026ndash;47.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-3\"\u003e\u0026uarr;\u003c/a\u003e Green, T., Toghill, A., Lee, R., Waechter, F., Weber, E., and Noakes, J. (2005a). Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 1: mode of action studies in the mouse. Toxicol. Sci. 86, 36\u0026ndash;47.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-4\"\u003e\u0026uarr;\u003c/a\u003e Pastoor, T., Rose, P., Lloyd, S., Peffer, R., and Green T. (2005). Thiamethoxam induced mouse liver tumors and their relevance to humans, Part 3: Weight of evidence evaluation of the human health relevance of thiamethoxam-related mouse liver tumors. Toxicological Sciences 86(1), 56\u0026ndash;60.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-5\"\u003e\u0026uarr;\u003c/a\u003e Carmichael, N. G., Enzmann, H., Pate, I., and Waechter, F. (1997). The significance of mouse liver tumor formation for carcinogenic risk assess- ment: Results and conclusions from a survey of ten years of testing. Environ. Health Perspect. 105, 1196\u0026ndash;1203.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-6\"\u003e\u0026uarr;\u003c/a\u003e Holsapple, M.P., Pitot, H.C., Cohen, S.H., Boobis, A.R., Klaunig, J.E., Pastoor, T., Dellarco, V.L., and Dragan, Y.P. (2006). Mode of Action in Relevance of Rodent Liver Tumors to Human Cancer Risk. Toxicological Sciences 89(1), 51\u0026ndash;56.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-7\"\u003e\u0026uarr;\u003c/a\u003e Bogdanffy, M.S. (2002). Vinyl acetate-induced intracellular acidification: implications for risk assessment. Toxicol Sci. 2002 Apr;66(2):320-6.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-8\"\u003e\u0026uarr;\u003c/a\u003e Meek, M., Bucher, J., Cohen, S., Dellarco, V., Hill, R., Lehman-McKeeman, L., Longfellow, D., Pastoor, T., Seed, J., and Patton, D. (2003). A Framework for human relevance analysis of information on carcinogenic modes of action. Crit. Rev. Toxicol. 33, 591\u0026ndash;653.\u003c/li\u003e\r\n\t\u003cli\u003e\u003ca href=\"#cite_ref-9\"\u003e\u0026uarr;\u003c/a\u003e Green, T., Toghill, A., Lee, R., Waechter, F., Weber, E., and Noakes, J. (2005a). Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 1: mode of action studies in the mouse. Toxicol. Sci. 86, 36\u0026ndash;47.\u003c/li\u003e\r\n\u003c/ol\u003e\r\n\r\n\u003ch2\u003eConfidence in the AOP\u003c/h2\u003e\r\n\r\n\u003cp\u003e\u003cspan style=\"color:red; font-size:200%\"\u003eInformation from this section should be moved to the Key Event Relationship pages!\u003c/span\u003e\u003cbr /\u003e\r\n\u0026nbsp;\u003c/p\u003e\r\n\r\n\u003cp\u003eThe coherence and extent of the database on thiamethoxam and its metabolites clearly demonstrates the mode of action for mouse liver tumorigenesis involving hepatocytotoxicity and regenerative cell proliferation \u003csup\u003e\u003ca href=\"#cite_note-10\"\u003e[1]\u003c/a\u003e\u003c/sup\u003e \u003csup\u003e\u003ca href=\"#cite_note-11\"\u003e[2]\u003c/a\u003e\u003c/sup\u003e The responses seen with thiamethoxam have been reproduced in studies of 50 and 20 weeks duration, the latter in two strains of mouse. The metabolite studies were internally consistent in that CGA330050 is only formed from thiamethoxam and not from the non-carcinogenic metabolite CGA322704. In all of the studies the key events had logical dose and temporal relationships. The metabolite studies were clear and consistent with the known carcinogenicity profiles of thiamethoxam and CGA322704. Studies on the metabolite CGA265307 and the inhibition of inducible nitric oxide synthase were limited, but showed that CGA265307 inhibits iNOS in vitro and enhances the toxicity of carbon tetrachloride in vivo. It is reasonable to conclude that CGA265307, although not toxic alone, could enhance the hepatotoxicity of metabolite CGA330050. Based on comparative metabolic studies neither rats nor humans would produce sufficient level of the two critical metabolites (CGA 330050 and CGA 265307) to initiate the progression of hepatic key events. As a consequence, it is unlikely that humans would be at risk of developing liver tumors as a result of exposure to thiamethoxam.\u003cbr /\u003e\r\n\u003cstrong\u003eCite error: \u003ccode\u003e\u0026lt;ref\u0026gt;\u003c/code\u003e tags exist, but no \u003ccode\u003e\u0026lt;references/\u0026gt;\u003c/code\u003e tag was found\u003c/strong\u003e\u003c/p\u003e\r\n","overall_assessment":"\u003cp\u003e\u003cem\u003eConsider the following criteria (may include references to KE Relationship pages): 1. concordance of dose-response relationships; 2. temporal concordance among the key events and adverse effect; 3. strength, consistency, and specificity of association of adverse effect and initiating event; 4. biological plausibility, coherence, and consistency of the experimental evidence; 5. alternative mechanisms that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP; 6. uncertainties, inconsistencies and data gaps. \u003c/em\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eThe AOP leading to thiamethoxam-induced hepatocarcinogenesis is a species-, time-, dose-, and metabolite-dependent process.\u003c/strong\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eDose and Temporal Concordance\u003c/strong\u003e In a 50-week mouse study with thiamethoxam, the earliest change, within one week, was a marked reduction (by up to 40%) in plasma cholesterol. This was followed 10 weeks later by evidence of liver toxicity including increased single-cell necrosis and increased apoptosis. After 20 weeks there was a significant increase in hepatic cell replication rates. All of these changes persisted from the time they were first observed until the end of the study at 50 weeks. Progression of events was consistently seen in several studies of 10, 20, or 50 weeks duration, with the hallmark indicator being a substantial decrease in plasma cholesterol levels \u003csup\u003e\u003ca href=\"#cite_note-1\"\u003e[1]\u003c/a\u003e\u003c/sup\u003e. The time-dependent key events occurs in a dose-response relationship that parallels the dose-related, late-life occurrence of tumors in mouse livers \u003csup\u003e\u003ca href=\"#cite_note-2\"\u003e[2]\u003c/a\u003e\u003c/sup\u003e and are only found at the carcinogenic dose (i.e., \u0026ge; 500 ppm).\u003c/p\u003e\r\n\r\n\u003cp\u003eThree metabolites (i.e., CGA 322704 and CGA 330050 which are further metabolize to CGA 265307) were identified and systematically evaluated for toxicological contribution to the sequence of hepatic effects. Mice and rats both produce CGA322704 as a major blood metabolite, which suggests that this particular metabolite is not an indicator of a species difference. However, CGA322704 does not cause liver tumors in mice nor does it cause any of the hepatic changes seen with thiamethoxam, and is thus considered not to be a part of causative chain of hepatic events. In contrast, CGA265307 and CGA330050 are produced in substantially greater quantity by mice than by rats (up to 140-fold and 15-fold greater, respectively), suggesting that the metabolic pathway through CGA330050 is critical to the AOP. In studies where these metabolites were fed to mice for at least ten weeks, CGA330050 was found to induce the same hepatic effects, and to the same degree, as thiamethoxam. CGA265307 alone induced none of the clinical or histopathological changes seen in the thiamethoxam-treated mice.\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eStrength, Consistency, Specificity of Association\u003c/strong\u003e All key events are well-defined measured effects with dose response and temporal concordance. The role of specific metabolites and time-dependent progression of hepatic lesions consistently seen including two strains of mice but not in rats. The role of CGA265307 was established by comparing its structural similarity to known inhibitors of inducible nitric oxide synthase (iNOS), by verifying the ability of CGA265307 to inhibit iNOS in vitro, and by assessing the ability of CGA265307 to exacerbate the iNOS-dependent hepatic toxicity of carbon tetrachloride in vivo. Based on structure-activity relationships and in vitro and in vivo experimentation, CGA265307\u0026rsquo;s role is thought to enhance the relatively mild hepatotoxicity induced by CGA330050, which leads to an increase in cellular death (via necrosis and apoptosis).\u003c/p\u003e\r\n\r\n\u003cp\u003eDifferences in metabolism between mice and rats, the contributory role of specific metabolites, and the time- dependent progression of hepatic lesions were consistently seen in a series of separate studies, including two strains of mice. \u003csup\u003e\u003ca href=\"#cite_note-3\"\u003e[3]\u003c/a\u003e\u003c/sup\u003e \u003csup\u003e\u003ca href=\"#cite_note-4\"\u003e[4]\u003c/a\u003e\u003c/sup\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003ePlausibility and Coherence\u003c/strong\u003e The phenomenon of a non-genotoxic mouse liver specific carcinogen is not uncommon in rodent bioassay studies. \u003csup\u003e\u003ca href=\"#cite_note-5\"\u003e[5]\u003c/a\u003e\u003c/sup\u003e Cytotoxicity and consequent regeneration is a well-known and well-documented mode of carcinogenic action for a variety of chemicals and for a variety of tissues including liver in laboratory animals. \u003csup\u003e\u003ca href=\"#cite_note-6\"\u003e[6]\u003c/a\u003e\u003c/sup\u003e \u003csup\u003e\u003ca href=\"#cite_note-7\"\u003e[7]\u003c/a\u003e\u003c/sup\u003e \u003csup\u003e\u003ca href=\"#cite_note-8\"\u003e[8]\u003c/a\u003e\u003c/sup\u003e\u003c/p\u003e\r\n\r\n\u003cp\u003e\u003cstrong\u003eAlternative modes of action\u003c/strong\u003e Genotoxicity, cytohrome P-450 induction, peroxisomal beta oxidation, and oxidative stress were considered experimentally and shown not to be viable.\u003csup\u003e\u003ca href=\"#cite_note-9\"\u003e[9]\u003c/a\u003e\u003c/sup\u003e\u003c/p\u003e\r\n","background":"","user_defined_mie":"","user_defined_ao":"","oecd_project":"","oecd_status_id":null,"graphical_representation_image_uid":"2016/11/29/773Liver_Tumors_from_Thiomethoxam.png","saaop_status_id":3,"legacy":true,"overall_assessment_file_uid":null,"changed_at":null,"development_strategy":null,"known_modulating_factors":null,"assigned_license_id":27,"handbook_id":1,"project_129":false}],"pagination":{"current_page":1,"per_page":25,"total_entries":578,"total_pages":24}}