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AOP Title

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Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits

Short name:

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Mitochondrial dysfunction and Neurotoxicity

Authors

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Anna Bal-Price, European Commission Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy

Marcel Leist, University of Konstanz, Konstanz, Germany

Stefan Schildknecht, University of Konstanz, Konstanz, Germany

Florianne Tschudi-Monnet, University of Lausanne and SCAHT, Lausanne, Switzerland

Alicia Paini, European Commission Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy

Andrea Terron, European Food Safety Authority, Parma Italy (corresponding author: Andrea.TERRON@efsa.europa.eu)

Point of Contact

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Andrea Terron

Contributors

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  • Andrea Terron
  • Clemens Wittwehr
  • Anna Price

Status

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Author status OECD status OECD project SAAOP status
Open for citation & comment EAGMST Under Review 1.33 Included in OECD Work Plan


This AOP was last modified on September 08, 2017 03:53

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Revision dates for related pages

Page Revision Date/Time
Inhibition, NADH-ubiquinone oxidoreductase (complex I) September 16, 2017 10:14
Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I) September 16, 2017 10:14
N/A, Mitochondrial dysfunction 1 September 16, 2017 10:14
Impaired, Proteostasis September 16, 2017 10:14
N/A, Neuroinflammation September 16, 2017 10:14
Degeneration of dopaminergic neurons of the nigrostriatal pathway September 16, 2017 10:14
Parkinsonian motor deficits November 30, 2016 15:24
N/A, Mitochondrial dysfunction 1 leads to Impaired, Proteostasis August 25, 2017 08:35
Impaired, Proteostasis leads to Degeneration of dopaminergic neurons of the nigrostriatal pathway August 25, 2017 08:37
N/A, Neuroinflammation leads to Degeneration of dopaminergic neurons of the nigrostriatal pathway August 25, 2017 08:54
Degeneration of dopaminergic neurons of the nigrostriatal pathway leads to N/A, Neuroinflammation August 25, 2017 09:21
N/A, Mitochondrial dysfunction 1 leads to Degeneration of dopaminergic neurons of the nigrostriatal pathway August 25, 2017 09:28
Degeneration of dopaminergic neurons of the nigrostriatal pathway leads to Parkinsonian motor deficits August 25, 2017 09:32
Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I) leads to Inhibition, NADH-ubiquinone oxidoreductase (complex I) August 25, 2017 09:35
Inhibition, NADH-ubiquinone oxidoreductase (complex I) leads to N/A, Mitochondrial dysfunction 1 August 25, 2017 09:43
1',2'-dihydrorotenone November 29, 2016 18:42
MPP+ December 16, 2016 11:22

Abstract

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This 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'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 α-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's disease (Keeney et al., 2006) to support the pathways of toxicity proposed in this AOP.


Background (optional)

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This optional section should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below.

Instructions

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Summary of the AOP

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Stressors

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Name Evidence Term
1',2'-dihydrorotenone Strong
MPP+ Strong

Molecular Initiating Event

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Title Short name
Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I) Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I)

Key Events

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Title Short name
Inhibition, NADH-ubiquinone oxidoreductase (complex I) Inhibition, NADH-ubiquinone oxidoreductase (complex I)
N/A, Mitochondrial dysfunction 1 N/A, Mitochondrial dysfunction 1
Impaired, Proteostasis Impaired, Proteostasis
N/A, Neuroinflammation N/A, Neuroinflammation
Degeneration of dopaminergic neurons of the nigrostriatal pathway Degeneration of dopaminergic neurons of the nigrostriatal pathway

Adverse Outcome

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Title Short name
Parkinsonian motor deficits Parkinsonian motor deficits

Relationships Between Two Key Events (Including MIEs and AOs)

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Network View

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Life Stage Applicability

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Life stage Evidence
Adult Strong

Taxonomic Applicability

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Term Scientific Term Evidence Link
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI

Sex Applicability

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Sex Evidence
Mixed Strong

Graphical Representation

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Click to download graphical representation template

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Overall Assessment of the AOP

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This section addresses the relevant domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and weight of evidence for the overall hypothesised AOP (i.e., including the MIE, KEs and AO) as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). It draws upon the evidence assembled for each KER as one of several components which contribute to relative confidence in supporting information for the entire hypothesised pathway. An important component in assessing confidence in supporting information as a basis to consider regulatory application of AOPs beyond that described in Section 6 is the essentiality of each of the key events as a component of the entire pathway. This is normally investigated in specifically-designed stop/reversibility studies or knockout models (i.e., those where a key event can be blocked or prevented). Assessment of the overall AOP also contributes to the identification of KEs for which confidence in the quantitative relationship with the AO is greatest (i.e., to facilitate determining the most sensitive predictor of the AO).

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Domain of Applicability

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This 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.


Essentiality of the Key Events

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Essentiality 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 α-synuclein) a KE show that such interference leads to an increase of KE down or the AO.

 

2 Support for Essentiality of KEs

Defining Question

Are downstream KEs and/or the AO prevented if an upstream KE is blocked ?

High (Strong)

Moderate

Low(Weak)

 

 

Direct 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.)

Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO

No or contradictory experimental evidence of the essentiality of any of the KEs

KE1

Inhibition of complex I

STRONG

Rationale: Inactivation of the NADH:Ubiquinone Oxidoreductase Core Subunit S7 (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 100 nM rotenone-mediated cell death (at 48 hrs of exposure) indicating that rotenone – induced toxicity requires rotenone 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).

 

KE2

Mitochondrial dysfunction

STRONG

Rationale: Many studies showing that antioxidants protect the cells against rotenone or MPTP 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+ 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+ induced neurotoxicity (Tieu K. et al. 2014).

KE3

Impaired proteostasis

MODERATE

Rationale: 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+ (Fornai et al. 2004; Dauer et al. 2002) . Injection of adeno/lenti-associated virus that expresses wild-type or mutant α-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).

Rationale 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)).

Rationale 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).

However, although many lines of evidence exist to support essentiality of impaired proteostasis, a single molecular chain of events cannot be established.

KE4

Degeneration of DA neurons of nigrostriatal pathway

MODERATE

Receptors 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+ neurons and mitigation of microglia and astrocytes reactivity was observed (Teismann et al. 2012). 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 (Sandström et al., 2017).

KE5

Neuroinflammation

MODERATE

Rationale: 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; ; 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+. 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).

KE 6

Degeneration of DA neurons of nigrostriatal pathway

STRONG

Rationale: 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).

Also, 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., 1998Silva 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).

Furthermore, 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).

 

 


Weight of Evidence Summary

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Concordance of dose-response relationship.

Data 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 µM 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’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 12-47µM) 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.

Table1 Dose-response and temporality table for rotenone.

 

Rotenone Concentration 

KE1aaa

Inhibition of C I

KE2aaa

Mitochondrial dysfunction

KE3aaa

Impaired proteostasis

KE4

Degeneration of DA neurons of nigrostriatal pathway

AO

Parkinsonian motor symptoms

5-10 nM in-vitro

[1]

+

4-72 hours [1]

+

4-72 hours [4]

+

24 hours [3]

-

-

20-30 nM ex-vivo, rat brain concentration

[4-5-2-6]

++

4-72 hours (4-5)

++

4-72 hours [4-5]

++

24 hours [3-2-6]

++a

5 weeks [2-6]

+++aa

5 weeks [2-6]

100 nM in-vitro

[4]

+++

4-72 hours [4]

+++

4-72 hours [4]

+++

24 hours [3]

Corresponding to a concentration above the maximum tolerated dose in-vivo [2-6]

Corresponding to a concentration above the maximum tolerated dose in vivo [2-6]

References: 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]

-no data available

+: low severity score, ++ intermediate severity score, +++ high severity score

a: 50% of treated animals showed loss of DA neurons in SNpc

aa: All animals affected in KE4 showed impaired motor symptoms

aaa: KE 1, 2 and 3 showed a dose-related severity in the effect and the score ++ was normalized vs. the KE4

Table 2. Dose-Response and Temporality table for MPTP/MPP+

MPTP Administered Dose

MPPBrain Concentration

KE1bb

Inhibition of C I

KE2bb

Mitochondrial dysfunction

KE3b

Impaired proteostasis

KE4

Degeneration of DA neurons of nigrostriatal pathway

AO

Parkinsonian motor symptoms

1 mg/kg sc infusion [1]

-

-

-

+

4 weeks[1]

+aaa

4 weeks [1]

No effect

5 mg/kg sc infusion [1]

-

-

-

++

4 weeks[1]

++aa

4 weeks [1]

+++

4 weeks [1]

20-30 mg/kg sporadic ip. injections (4 times every 2 hours)

[2, 1]

 

47µM [2]^

12µM [1]

+++

4 hrs [2]

+++

4hrs [2]

+++

4 weeks [1]

+++a

1-4 weeks[2,1]

 

+++

4 weeks [1]


References. Fornai et al. 2005 [1]; Thomas et al. 2012 [2]

-no data available

a: approx 50% loss of DA neurons in SNpc

aa: approx 30% loss of DA neurons SN pc

aaa: no loss of DA neurons in SN pc. Reduced level of striata DA

b: for KE3, a dose response effect was observed.

bb: for KE 1 and 2 the severity of the effect was normalized vs. the KE4

^ After single dose MPTP administration, brain concentration was approx. 5.15 µM

Temporal concordance among the MIE, KEs and AO.

There 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’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).

Strength, consistency, and specificity of association of AO and MIE.

Strength 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  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; therefore, kinetic and metabolic considerations should be taken into account to fully 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:

The 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.

All of these structurally different complex I inhibitors were characterized with isolated mitochondria or with submitochondrial particles. Application of bovine heart mitochondria revealed IC50 values in the range of 20-70 nM for piericidin A, fenpyroximate, rotenone, and phenoxan (Eur. J. Biochem 1994, 219, 691-698). IC50 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).

Studies involving neuronal cell cultures or in vivo models are in fact rather rare. A systematic comparison of the IC50 values for complex I inhibition and EC50 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 EC50 values were in most cases lower than the IC50 values detected for complex I inhibition. EC50 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).

A 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 EC50 values of 20 nM (annonacin), 34 nM (rotenone), and 1900 nM (MPP+) (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)).

 

Weight of Evidence (WoE).

Biological plausibility, coherence, and consistency of the experimental evidence.

The 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).

 

1 Support for Biological Plausibility of KERs

Defining Question

High (Strong)

Moderate

Low(Weak)

 

Is there a mechanistic (i.e. structural or functional) relationship between KEup and KE down consistent with established biological knowledge?

Extensive understanding of the KER based on extensive previous documentation and broad acceptance

The KER is plausible based on analogy to accepted biological relationships, but scientific understanding is not completely established

There is empirical support for a statistical association between KEs but the structural or functional relationship between them is not understood

MIE => KE1

Binding of inhibitor to NADH-ubiquinone oxidoreductase leads of complex I

STRONG

Rationale: 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).

KE1 => KE2

Inhibition of complex I leads to mitochondrial dysfunction

STRONG

Rationale: 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

KE2 => KE3

Mitochondrial dysfunction results in impaired proteostasis

MODERATE

Rationale: 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).

KE2 => KE4

Mitochondrial dysfunction leads to the degeneration of dopaminergic neurons of the nigrostriatal pathway

 

STRONG

Rationale: 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).

KE3 => KE4

Impaired proteostasis leads to degeneration of DA neurons of the nigrostriatal pathway

MODERATE

Rationale: 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.

KE4 => KE5

Degeneration of DA neurons of the nigrostriatal pathway leads to neuroinflammation

MODERATE

Rationale: 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).

KE5 => KE6

Neuroinflammation leads to degeneration of DA neurons of the nigrostriatal pathway

MODERATE

Rationale: 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 ; Brown and Bal-Price, 2003 ; Kraft and Harry, 2011 ; Taetzsch and Block, 2013). However, the responsible mediators differ from model to model.

KE6 => AO

Degeneration of DA neurons of the nigrostriatal pathway leads to parkinsonian motor symptoms

STRONG

Rationale: 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 % of striatal DA is depleted as a consequence of SNpc DA neuronal degeneration (Koller et al. 1992).

Empirical support.

Empirical support is strong. Many studies show evidence for the KERs by showing temporal concordance and dose concordance when using different stressors.

 

3 Empirical support for KERs

Defining Question

Does 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?

Are inconsistencies in empirical support cross taxa, species and stressors that don’t align with expected pattern of hypothesized AOP?

High (Strong)

Moderate

Low(Weak)

 

 

Multiple 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.

Demonstrated 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.

Limited 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’t align with expected pattern for hypothesized AOP

MIE => KE1

Binding of inhibitor to NADH-ubiquinone oxidoreductase leads to partial or total inhibition of complex I

STRONG

Rationale: 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).

KE1 => KE2

Inhibition of complex I leads to mitochondrial dysfunction

STRONG

Rationale: 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).

KE2 => KE3

Mitochondrial dysfunction results in impaired proteostasis

STRONG

Rationale: 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 α-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).

KE2 => KE4

 

Mitochondrial dysfunction directly leads to degeneration of DA neurons of nigrostriatal pathway

 

STRONG

Rationale: Multiple in vitro studies indicate dose and response-response concordance. As most of the studies were conducted in vitro, 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)

KE3 => KE4

Impaired proteostasis leads to degeneration of DA neurons of the nigrostriatal pathway

STRONG

Rationale: 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).

KE4 => KE5

Degeneration of DA neurons of nigrostriatal pathway leads to neuroinflammation

MODERATE

Rationale: 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.

KE5 => KE6

Neuroinflammation leads to degeneration of DA neurons of nigrostriatal pathway parkinsonian motor symptoms

MODERATE

Rationale: 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).

KE6 => AO

Degeneration of DA neurons of nigrostriatal pathway leads to parkinsonian motor symptoms

STRONG

Rationale: 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 ie 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).

Uncertainties and Inconsistencies.

  • The 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)  or produce a similar AO.
  • Conflicting 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, challenging the current AOP. The cited research article shows that abolishment of complex I’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. 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 a minor inconsistency. 
  • There 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.
  • It 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).
  • Some studies suggest that rotenone and MPTP/MPP+ may have effects other than CI inhibition, e.g. MPTP and rotenone can induce microtubule disruption (Feng, 2006; Ren et al., 2005Cappelletti et al., 1999; Cappelletti et al., 2001, Brinkley et al., 1974; Aguilar et al. 2015).
  • There are additional feedback possible between KEs, e.g. ROS production from KE2 may damage CI, this leads to enhancement of KE1.
  • Some KEs e.g. KE 2, 3, 5 pool molecular processes that may need to be evaluated individually at a later stage.
  • The exact molecular link from mitochondrial dysfunction to disturbed proteostasis is still unclear (Malkus et al 2009; Zaltieri et al. 2015).
  • The role of ATP depletion vs. other features of mitochondrial dysfunction is not clear.
  • The role of a α-synuclein in neuronal degeneration is still unclear as well as the mechanisms leading to its aggregation.
  • It is not clear under which conditions KE3 and KE5 become modulatory factors, and when they are essential. MPTP/MPP+ 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+ 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.
  • Inconsistent 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).
  • The selective vulnerability of the SN pc dopaminergic pathway does not have a molecular explanation.
  • Priority 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’s result and extrapolation of potential low-dose chronic effect as this AOP refers to long-time exposure.
  • The 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 o PD symptoms is also representing an uncertainties.
  • There 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.
  • This 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).

 


Quantitative Considerations

?

The 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 µM 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 “threshold”. 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.


Table 3 Summary of quantitative effects at the concentration of rotenone and MPTP triggering the AO.

Concentration

KE1

Inhibition of C I

KE2

Mitochondrial dysfunction

KE3

Impaired proteostasis

KE4

Degeneration of DA neurons of nigrostriatal pathway

AO

Parkinsonian motor symptoms

Rotenone 20-30 nM rat brain concentration

[1-2]

Approx. 53%[4-5]

Approx. 20-53% (decrease in respiration rate)[1-2]

Approx. 20-60% (decrease in UPS (26S) activity) [3]

Neuronal loss (50% of animal affected) [2]

Motor impairment (100% of animals with neuronal loss) [2]

MPP+ 12-47 µM rat brain concentration [4-5]

Approx. 50-75% [5]

Approx. 38% (reduction in phosphorylating respiration) [5]

Approx. 60% (decrease in UPS activity) [4]

Approx. 50% of neuronal loss [4-5]

Motor impairment [4]


[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.


Summary of the proposed Key Events in this AOP:

Final graph.jpg

Chronic, 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 α-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'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.


Considerations for Potential Applications of the AOP (optional)

?


  1. This 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 studiesto 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.
  2. In 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.
  3. The 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.
  4. This 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.

  5. The 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 in vitro can be extrapolated to in vivo and exposure scenarios can be simulated.

  6. This 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.

 


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?


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