<p>Katherine von Stackelberg <span style="font-size:12px"><em>(Harvard Center for Risk Analysis, Boston, MA, USA.) (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)</em></span></p>
<p>Elizabeth Guzy <em><span style="font-size:12px">(Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)</span></em></p>
<p>Tian Chu <span style="font-size:12px"><em>(Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)</em></span></p>
<p>Birgit Claus Henn <span style="font-size:12px"><em>(Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.) (Department of Environmental Health, Boston University School of Public Health, Boston, MA, USA.)</em></span></p>
<p><u>Of the content populated in the AOP-Wiki:</u></p>
<p>Travis Karschnik <span style="font-size:12px"><em>(General Dynamics Information Technology, Duluth, MN, USA.)</em></span></p>
<td>Under development: Not open for comment. Do not cite</td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
<h2>Abstract</h2>
<p>Metal mixture activation of ERK1/2 and JNK1/2 in astrocytes leads to increased Ca2+ release (Asit Rai et al., 2010). Alterations to calcium, an essential nutrient which is required in multiple cellular and physiological functions, such as cell adhesion, signal transduction, and neurotransmission can be expected to have downstream effects in those functions (Antonio et al., 2002). While a variety of stimuli can trigger opening of the mitochondrial transition pore and cause apoptosis, a sustained intracellular increase in Ca2+ is one of the better-known triggers (Mattson 2000). Mitochondria play a role in stress responses and can produce ROS when damaged. Mitochondria are indeed a major source of ROS (Yan et al., 2013). Unchecked, excessive ROS can lead to the destruction of cellular components including lipids, protein, and DNA, and ultimately cell death via apoptosis or necrosis (Kannan and Jain 2000). Aberrant apoptosis has been implicated in the pathogenesis of neurodegeneration (Okouchi et al., 2007). It is well accepted that impairment of cell function or cell loss (neurodegeneration) in hippocampus will interfere with memory processes, since the hippocampus plays a key role in memory (Barker and Warburton 2011).</p>
<p>MEK-ERK1/2 is important in understanding uptake of metals into the brain and its relationship to deficits in learning and cognition from exposure to metals commonly detected at Superfund sites including lead, cadmium, manganese, and arsenic. Current risk assessment guidance dictates a largely chemical-by-chemical evaluation of exposures and risks, which fails to adequately address potential interactions with other chemicals, nonchemical stressors, and genetic factors. Cumulative risk assessment methods and approaches are evolving to meet regulatory needs (MacDonell et al., 2013; Backhaus and Faust 2012; IPCS 2009), but significant challenges remain. As our understanding of complex exposures and interactions continues to grow, synthesis and integration across disciplines and studies focused on different aspects of the environmental fate–exposure–toxicology–health outcome continuum are required to assess the likelihood of adverse effects and to support cumulative risk assessment. Environmental exposures are virtually always to complex mixtures (von Stackelberg et al., 2015).</p>
<p> </p>
</div>
<div id="background">
<h3>Background</h3>
<p>An examination of neurodevelopmental disorders and subclinical effects using multi-domain global neurodevelopment assessments is warranted as they can have profound population level implications. In the context of neurotoxicity, neurodevelopmental pathways in the developing human brain are not fully understood (Schubert et al., 2013; Bal-Price et al., 2015) although there are a number of commonly observed phenomena which may take part in those pathways e.g. changes in intracellular calcium, ROS generation, apoptosis, and neurotransmitter disruption. This AOP highlights a specific set of response-response relationships using a subset of those commonly observed phenonema related to metals and metal mixture exposures leading to deficits in learning and cognition.</p>
<div>
<p>This AOP was developed as part of an Environmental Protection Agency effort to increase the impact of AOPs published in the peer-reviewed literature, but heretofore unrepresented in the AOP-Wiki, by facilitating their entry and update. The originating work for this AOP was <strong>Katherine von Stackelberg & Elizabeth Guzy & Tian Chu & Birgit Claus Henn, 2015. Exposure to Mixtures of Metals and Neurodevelopmental Outcomes: A Multidisciplinary Review Using an Adverse Outcome Pathway Framework, Risk Analysis, John Wiley & Sons, vol. 35(6), pages 971-1016, June.</strong> This publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages.</p>
<p>An examination of neurodevelopmental disorders and subclinical effects using multi-domain global neurodevelopment assessments is warranted as they can have profound population level implications. In the context of neurotoxicity, neurodevelopmental pathways in the developing human brain are not fully understood(Schubert et al., 2015; Bal-Price et al., 2015) although there are a number of commonly observed phenomena which may take part in those pathways e.g. changes in intracellular calcium, ROS generation, apoptosis, and neurotransmitter disruption. This AOP highlights a specific set of response-response relationships using a subset of those commonly observed phenonema related to metals and metal mixture exposures leading to deficits in learning and cognition. </p>
<p>The focus of the originating work was to conduct a review of the literature on relationships between prenatal and early life exposure to mixtures of lead (Pb), arsenic (As), cadmium (Cd), and manganese (Mn) with neurodevelopmental outcomes and then use an AOP framework to integrate lines of evidence from multiple disciplines based on evolving guidance developed by the Organization for Economic Cooperation and Development (OECD). Importantly, the review considered whether exposures to mixtures of metals was associated with neurodevelopment effects that were greater or less than effects from exposure to each individual metal.</p>
<p><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><strong><span style="font-size:8.0pt">1. Support for Biological Plausibility of KERs</span></strong></span></span></p>
<p style="margin-left:14px; margin-right:20px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Is there a mechanistic relationship between KE</span><span style="font-size:5.0pt">up </span><span style="font-size:8.0pt">and KE</span><span style="font-size:5.0pt">down </span><span style="font-size:8.0pt">consistent with established biological knowledge?</span></span></span></p>
<p style="margin-left:14px; margin-right:6px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Extensive</span> <span style="font-size:8.0pt">understanding of the KER based on extensive previous documentation and broad acceptance.</span></span></span></p>
<p style="margin-left:14px; margin-right:10px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete</span></span></span></p>
<p style="margin-left:14px; margin-right:18px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Empirical support for association between KEs , but the structural or functional relationship between them is not understood.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Empirical evidence indicates a complex relationship between MEK, ERK1/2 activation and inhibition and Ca2+ response including Ca2+ feeding back into a ERK1/2 activation. This relationship appears to vary across species and cell type.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">There are both accepted associations between these two KEs and empirical evidence but the current state of understanding falls short of extensive.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">This relationship has been studied in humans and human-model rodents extensively related to age-related diseases.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">This is a well-studied relationship across taxa where modulation of ROS and its effect on subsequent apoptosis has been examined.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">This relationship has been studied in humans and human-model rodents extensively related to age-related diseases.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">This relationship has been studied in humans and human-model rodents extensively related to age-related diseases.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">This relationship has been studied in humans and human-model rodents extensively related to age-related diseases.</span></span></span></p>
<p>Life stages applicable to this AOP encompass the full life cycle. Many of the key events are measured in pregnant females with the adverse outcome (impairment, learning and memory) measured at all life stages.</p>
<p><strong>Taxonomic Applicability</strong></p>
<p>Most evidence for this AOP is derived from rodents and humans where rodents were selected with their ability to model human responses.</p>
<p style="margin-left:7px; margin-right:6px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</span></span></span></p>
<p style="margin-left:7px; margin-right:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs</span></span></span></p>
<p style="margin-left:7px; margin-right:9px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE</span></span></span></p>
<p style="margin-left:7px; margin-right:8px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">No or contradictory experimental evidence of the essentiality of any of the KEs.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">MEK, ERK1/2 activation is fundamental in delivering signals which regulate the cell cycle, proliferation, differentiation, adhesion, and more. Disruptions in this activation have wide reaching effects however, there is evidence that downstream KEs can also activate this KE. </span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Calcium, as a primary intracellular messenger in neurons and regulator of cell responses to stress has been shown to play an integral role in subsequent KEs.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The ubiquity and role of mitochondria in cell function is such that changes in this KE necessitate changes in downstream KEs.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">ROS has been shown to mediate apoptosis across taxa with changes in ROS levels affecting subsequent apoptosis.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Unregulated apoptosis has been shown to affect neurodegeneration and eventual learning and memory tasks in human and rodent models.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Neurodegeneration has been causally linked to learning and memory tasks in human and rodent models.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">There is evidence for manipulation of downstream KEs based on manipulation of upstream KEs in multiple KERs.</span></span></span></p>
<p style="margin-left:14px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><strong><span style="font-size:8.0pt">3. Empirical Support for KERs</span></strong></span></span></p>
<p style="margin-left:14px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Does 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 > than that for KEdown? Inconsistencies?</span></span></span></p>
<p style="margin-left:14px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">if there is dependent change in both events following exposure to a wide range of specific stressors (extensive evidence for temporal, dose- response and incidence concordance) and no or few data gaps or conflicting data</span></span></span></p>
<p style="margin-left:14px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">if there is demonstrated</span> <span style="font-size:8.0pt">dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with the expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories,</span> <span style="font-size:8.0pt">etc.</span></span></span></p>
<p style="margin-left:14px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">if there are limited or no studies reporting dependent</span> <span style="font-size:8.0pt">change in both events following exposure to a specific stressor (i.e., endpoints never measured in the same study or not at all), and/or lacking evidence of temporal or dose- response concordance, </span><span style="font-size:8.0pt">or identification</span> <span style="font-size:8.0pt">of significant inconsistencies</span> <span style="font-size:8.0pt">in empirical support across taxa and species that don’t align with the expected pattern for the hypothesised</span> <span style="font-size:8.0pt">AOP </span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">T</span></span></span><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">he evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure. Heavy metals like cadmium can complicate issues related to calcium levels since the metal itself can act in place of calcium in cell function.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures. Some inconsistency was documented in the relationship between the two events regarding which preceded the other in different taxa and cell types. Heavy metals like cadmium can complicate issues related to calcium levels since the metal itself can act in place of calcium in cell function.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure. There are also numerous studies investigating this relationship in the context of neurodegenerative diseases.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure. There are also numerous studies investigating this relationship in the context of neurodegenerative diseases.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.</span></span></span></p>
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<p>Developmental neurotoxicity (DNT) is an adverse outcome of concern to multiple regulatory agencies. In vitro screening assays for MEK-ERK1/2 activation would not be recommended as a direct alternative or replacement to established DNT assays like OECD Test No. 426 (OECD 2007). However, detection of MEK-ERK1/2 activation in neuronal cell types may be used to prioritize chemicals with potential to elicit neurotoxicity and flag them for testing in ortogonal assays for evaluating DNT, including proposed alternative test methods (Bal-Price et al. 2018; Crofton et al 2022). </p>
</div>
<div id="references">
<h2>References</h2>
<p>Asit Rai and others, Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601, https://doi.org/10.1093/toxsci/kfq266</p>
<p>Backhaus T, Faust M. Predictive environmental risk assessment of chemical mixtures: A conceptual framework. Environmental Science & Technology, 2012; 46(5):2564–2573.</p>
<p>Bal-Price A, Crofton KM, Sachana M, Shafer TJ, Behl M, Forsby A, Hargreaves A, Landesmann B, Lein PJ, Louisse J, Monnet-Tschudi F, Paini A, Rolaki A, Schrattenholz A, Sunol C, van Thriel C, Whelan M, Fritsche E. Putative adverse outcome pathways relevant to neurotoxicity. Critical Reviews in Toxicology, 2015; 45(1):83–91.</p>
<p>Bal-Price A, Hogberg HT, Crofton KM, Daneshian M, FitzGerald RE, Fritsche E, Heinonen T, Hougaard Bennekou S, Klima S, Piersma AH, Sachana M, Shafer TJ, Terron A, Monnet-Tschudi F, Viviani B, Waldmann T, Westerink RHS, Wilks MF, Witters H, Zurich MG, Leist M. Recommendation on test readiness criteria for new approach methods in toxicology: Exemplified for developmental neurotoxicity. ALTEX. 2018;35(3):306-352. doi: 10.14573/altex.1712081. Erratum in: ALTEX. 2019;36(3):506. </p>
<p>Barker GR, Warburton EC. 2011. When is the hippocampus involved in recognition memory? J Neurosci 31(29): 10721-10731.</p>
<p>Crofton KM, Bassan A, Behl M, Chushak YG, Fritsche E, Gearhart JM, Marty MS, Mumtaz M, Pavan M, Ruiz P, Sachana M, Selvam R, Shafer TJ, Stavitskaya L, Szabo DT, Szabo ST, Tice RR, Wilson D, Woolley D, Myatt GJ. Current status and future directions for a neurotoxicity hazard assessment framework that integrates <em>in silico</em> approaches. Comput Toxicol. 2022 May;22:100223. doi: 10.1016/j.comtox.2022.100223. </p>
<p>International Programme on Chemical Safety (IPCS),World Health Organization (WHO). Assessment of combined exposures to multiple chemicals. Report of a WHO/IPCS International Workshop, 2009.</p>
<p>Katherine von Stackelberg & Elizabeth Guzy & Tian Chu & Birgit Claus Henn, 2015. "Exposure to Mixtures of Metals and Neurodevelopmental Outcomes: A Multidisciplinary Review Using an Adverse Outcome Pathway Framework," Risk Analysis, John Wiley & Sons, vol. 35(6), pages 971-1016, June.</p>
<p>M.Teresa Antonio, Noelia López, M.Luisa Leret, Pb and Cd poisoning during development alters cerebellar and striatal function in rats, Toxicology, Volume 176, Issues 1–2, 2002,<br />
<p>MacDonell MM, Haroun LA, Teuschler LK, Rice GE, Hertzberg RC, Butler JP, Chang Y-S, Clark SL, John AP, Perry CS, Garcia SS, Jacob JH, Scofield MA. 2013. Cumulative risk assessment toolbox:Methods and approaches for the practitioner. Journal of Toxicology, 2013; Article ID 310904, doi:10.1155/2013/310904.</p>
<p>Mattson, M. Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1, 120–130 (2000). https://doi.org/10.1038/35040009</p>
<p>OECD (2007), <em>Test No. 426: Developmental Neurotoxicity Study</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264067394-en">https://doi.org/10.1787/9789264067394-en</a>.</p>
<p>Okouchi, Masahiro, et al. "Neuronal apoptosis in neurodegeneration." Antioxidants & redox signaling 9.8 (2007): 1059-1096.</p>
<p>Schubert D, Martens GJM, Kolk SM. Molecular underpinnings of prefrontal cortex development in rodents provide insights into the etiology of neurodevelopmental disorders. Molecular Psychiatry, 2013; 2014:1–15.</p>
<p>Yuan, Yan, et al. "Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway." PloS one 8.5 (2013): e64330.</p>
</div>
<div id="appendicies">
<h2>Appendix 1</h2>
<h3>List of MIEs in this AOP</h3>
<h4><a href="/events/2146">Event: 2146: Activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase 1/2</a></h4>
<p>ERK1 and ERK2 are proteins of 43 and 41 kDa that are nearly 85% identical overall, with much greater identity in the core regions involved in binding substrates (Boulton et al., 1990; 1991). The two phosphoacceptor sites, tyrosine and threonine, which are phosphorylated to activate the kinases, are separated by a glutamate residue in both ERK1 and ERK2 to give the motif TEY in the activation loop (Payne et al., 1991). Both are ubiquitously expressed, although their relative abundance in tissues is variable. For example, in many immune cells ERK2 is the predominant species, while in several cells of neuroendocrine origin they may be equally expressed (Gray Pearson and others 2001). They are stimulated to some extent by a vast number of ligands and cellular perturbations, with some cell type specificity (Lewis et al., 1998). In fibroblasts (the cell type in which the generalizations about their behavior and functions have been developed) they are activated by serum, growth factors, cytokines, certain stresses, ligands for G protein-coupled receptors (GPCRs), and transforming agents, to name a few (Gray Pearson and others 2001). They are highly expressed in postmitotic neurons and other highly differentiated cells (Boulton et al., 1991). In these cells they are often involved in adaptive responses such as long-term potentiation (English and Sweatt 1996; Atkins et al., 1998; Rossi-Arnaud et al., 1997).</p>
<h4>How it is Measured or Detected</h4>
<p>Western blotting and immunoblotting.</p>
<h4>References</h4>
<p>Atkins CM, Selcher JC, Petraitis JJ, Trzaskos JM, Sweatt JD 1998 The MAPK cascade is required for mammalian associative learning. Nat Neurosci 1 :602 –609</p>
<p>Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, DePinho RA, Panayotatos N, Cobb MH, Yancopoulos GD 1991 ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65 :663 –675</p>
<p>Boulton TG, Yancopoulos GD, Gregory JS, Slaughter C, Moomaw C, Hsu J, Cobb MH 1990 An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 249 :64 –67</p>
<p>English JD , Sweatt JD 1996 Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem 271 :24329 –24332</p>
<p>Gray Pearson and others, Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological Functions, Endocrine Reviews, Volume 22, Issue 2, 1 April 2001, Pages 153–183, https://doi.org/10.1210/edrv.22.2.0428</p>
<p>Lewis TS, Shapiro PS, Ahn NG 1998 Signal transduction through MAP kinase cascades. Adv Cancer Res 74 :49 –139</p>
<p>Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J-H, Shananowitz J, Hunt DF, Weber MJ, Sturgill TW 1991 Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J 10 :885 –892</p>
<p>Rossi-Arnaud C, Grant SG, Chapman PF, Lipp HP, Sturani E, Klein R 1997 A role for the Ras signalling pathway in synaptic transmission and long-term memory. Nature 390 :281 –286</p>
<p>Calcium is arguably the most versatile and important intracellular messenger in neurons<span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span>(Berridge et al., 2000). Interestingly, although calcium may often promote neuronal death, it can also activate pathways that promote survival. For example, calcium can promote survival through a pathway involving activation of protein kinase B (PKB/Akt) by calcium/calmodulin-dependent protein kinase (Yano et al., 1998). Calcium is a prominent regulator of cellular responses to stress, activating transcription through the cyclic-AMP response element-binding protein (CREB), which can promote neuron survival in experimental models of developmental cell death (Hu et al., 1999). Calcium can also activate a rapid neuroprotective signalling pathway in which the calcium-activated actin-severing protein gelsolin induces actin depolymerization, resulting in suppression of calcium influx through membrane NMDA (N-methyl-d-aspartate) receptors and voltage-dependent calcium channels (Furukawa et al., 1997). This may occur through intermediary actin-binding proteins that interact with NMDA receptor and calcium channel proteins. Finally, signals such as calcium and secreted amyloid precursor protein-α (sAPP-α), which increase cyclic GMP production, can induce activation of potassium channels and the transcription factor NF-κB, and thereby increase resistance of neurons to excitotoxic apoptosis (Furukawa et al., 1996).</p>
<h4>How it is Measured or Detected</h4>
<p>An increase in [Ca<sup>2+]</sup>i was measured using Fluo3 AM as an indicator dye after the addition of metals (single or in mixture) to the culture wells following an optimized protocol (Arey et al., 2022). The fluorescent signals were read by fluorescence imaging plate reader Synergy HT (BioTek, Winooski, VT) (Rai and others 2010).</p>
<p>Briefly, Ca2+ levels in human astrocytes were monitored by fluorescence microscopy using the Ca2+ indicator fluo-4. Slices were incubated with fluo-4-AM (2–5 µL of 2 mM dye were dropped over the tissue, attaining a final concentration of 2–10 µM and 0.01% of pluronic) and Sulforhodamine 101 (100 µM) for 30–60 min at room temperature (Navarrete and others 2013). In these conditions, most of the Fluo-4-loaded cells were astrocytes as indicated by their SR101 staining (Nimmerjahn et al., 2004; Dombeck et al., 2007; Kafitz et al., 2008; Takata and Hirase 2008), and confirmed in some cases by their electrophysiological properties. Astrocytes were imaged with an Olympus FV300 laser-scanning confocal microscope or a CCD camera (Retiga EX) attached to the Olympus BX50WI microscope (Navarrete and others 2013).</p>
<p>Diversity of endogenous Ca2+ activity in a mature hippocampal astrocyte in situ: Ca2+ signals in cell body and processes are different. (A) Cumulative Ca2+ activity recorded in an astrocyte over a 165 s period revealed by the calcium indicator Fluo4-AM. The visible boundaries of the astrocyte are shown in white. Note the different intensities of spatially-<br />
confined local activity in the astrocyte cell body (s), primary process (p1) stemming from the soma and secondary processes (p2) branching from a primary process. Intensity of the<br />
normalized cumulative activity is expressed in arbitrary units (a.u.) and shown in pseudocolour, from dark (lowest) to white (highest). (B) Frequency map of the Ca2+ activity in the astrocyte during the 165 s period as in A. Activity is measured in individual pixels, expressed in mHz and color-coded from black (never active) to dark red (frequently active). Most of the activity is within the white boundaries and the most frequently active pixels are in defined small regions (arrowheads) of the primary and secondary processes (30 mHz), whereas pixels of the soma are less active (~10 mHz) (Volterra et al., 2014). </p>
<p>Free intracellular calcium ions were measured using the fluorescent calcium indicator FLUO-3/AM (Molecular probes, Eugene, OR, USA). Cells (4 × 10<sup>4</sup> cells/cm<sup>2</sup>) were seeded in 24-well plates for 24 h to reach 60%–70%, and then treated for 24 h with As(III) (0.5 and 1 mg/l), or coexposed to As(III) (1 mg/l) and F (2.5, 5, and 10 mg/l). After treatment, supernatant was collected and combined with trypsinized cells. Pelleted samples were resuspended in 500 μl of FLUO-3/AM (4 μmol/l) and incubated at 37 °C for 30 min. After centrifugation, cells were washed with HBSS (Hank's Buffered Salt Solution, Sigma), made up to 400 μl with HBSS and analyzed by flow cytometry. The signal from FLUO-3/AM bound to Ca<sup>2+</sup> was recorded using the Fl-1 channel (Rocha et al., 2011).</p>
<p>Fluo-4/AM was used as an intracellular free Ca<sup>2+</sup> fluorescent probe to analyze [Ca<sup>2+</sup>]<sub>i</sub> in Cd-exposed cerebral cortical neurons. In short, the harvested cells were incubated with Fluo-4/AM (5 µmol/L final concentration) for 30 min at 37°C in the dark, washed with PBS, and analyzed on a BD-FACS Aria flow cytometry. Intracellular [Ca<sup>2+</sup>]<sub>i</sub> levels were represented by fluorescent intensity. Fluorescent intensity was recorded by excitation at 494 nm and emission at 516 nm. The data were analyzed by Cell Quest program (Becton Dickinson), and the mean fluorescence intensity was obtained by histogram statistics (Yuan et al., 2013).</p>
<h4>References</h4>
<p>Arey BJ Seethala R Ma Z Fura A Morin J Swartz J Vyas V Yang W Dickson JK JrFeyen JH A novel calcium-sensing receptor antagonist transiently stimulates parathyroid hormone secretion in vivo Endocrinology 2005 146 2015 2022</p>
<p>Asit Rai and others, Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601, https://doi.org/10.1093/toxsci/kfq266</p>
<p>Berridge, M. J., Lipp, P. & Bootman, M. D. The versatility and universality of calcium signaling. Nature Rev. Mol. Cell Biol. 1, 11– 21 (2000).</p>
<p>Dombeck DA, Khabbaz AN, Collman F, Adelman TL, Tank DW. Imaging large-scale neural activity with cellular resolution in awake, mobile mice, Neuron, 2007, vol. 56 (pg. 43-57)</p>
<p>Furukawa, K. et al. The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J. Neurosci. 17, 8178– 8186 (1997).</p>
<p>Furukawa, K., Barger, S. W., Blalock, E. M. & Mattson, M. P. Activation of K+ channels and suppression of neuronal activity by secreted β-amyloid-precursor protein. Nature 379, 74–78 (1996).</p>
<p>Hu, S. C., Chrivia, J. & Ghosh, A. Regulation of CBP-mediated transcription by neuronal calcium signaling. Neuron 22, 799– 808 (1999).</p>
<p>Kafitz KW, Meier SD, Stephan J, Rose CR. Developmental profile and properties of sulforhodamine 101-labeled glial cells in acute brain slices of rat hippocampus, J Neurosci Methods, 2008, vol. 169 (pg. 84-92)</p>
<p>Marta Navarrete and others, Astrocyte Calcium Signal and Gliotransmission in Human Brain Tissue, Cerebral Cortex, Volume 23, Issue 5, May 2013, Pages 1240–1246, https://doi.org/10.1093/cercor/bhs122</p>
<p>Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo, Nat Methods, 2004, vol. 1 (pg. 31-37)</p>
<p>R.A. Rocha, J.V. Gimeno-Alcañiz, R. Martín-Ibañez, J.M. Canals, D. Vélez, V. Devesa, Arsenic and fluoride induce neural progenitor cell apoptosis, Toxicology Letters, Volume 203, Issue 3, 2011, Pages 237-244, ISSN 0378-4274, https://doi.org/10.1016/j.toxlet.2011.03.023.</p>
<p>Takata N, Hirase H. Cortical layer 1 and layer 2/3 astrocytes exhibit distinct calcium dynamics in vivo., PLoS ONE, 2008, vol. 3 pg. e2525</p>
<p>Volterra, Andrea, Nicolas Liaudet, and Iaroslav Savtchouk. "Astrocyte Ca2+ signalling: an unexpected complexity." Nature Reviews Neuroscience 15.5 (2014): 327-335.</p>
<p>Yano, S., Tokumitsu, H. & Soderling, T. R. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature 396, 584–587 (1998).</p>
<p>Yuan Y, Jiang C-y, Xu H, Sun Y, Hu F-f, Bian J-c, et al. (2013) Cadmium-Induced Apoptosis in Primary Rat Cerebral Cortical Neurons Culture Is Mediated by a Calcium Signaling Pathway. PLoS ONE 8(5): e64330. https://doi.org/10.1371/journal.pone.0064330</p>
<td><a href="/aops/48">Aop:48 - Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.</a></td>
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<td><a href="/aops/77">Aop:77 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony death/failure 1</a></td>
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<td><a href="/aops/78">Aop:78 - Nicotinic acetylcholine receptor activation contributes to abnormal role change within the worker bee caste leading to colony death failure 1</a></td>
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<td><a href="/aops/79">Aop:79 - Nicotinic acetylcholine receptor activation contributes to impaired hive thermoregulation and leads to colony loss/failure</a></td>
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<td><a href="/aops/80">Aop:80 - Nicotinic acetylcholine receptor activation contributes to accumulation of damaged mitochondrial DNA and leads to colony loss/failure</a></td>
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<td><a href="/aops/87">Aop:87 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony loss/failure </a></td>
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<td><a href="/aops/3">Aop:3 - Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits</a></td>
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<td><a href="/aops/144">Aop:144 - Endocytic lysosomal uptake leading to liver fibrosis</a></td>
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<td><a href="/aops/178">Aop:178 - Nicotinic acetylcholine receptor activation contributes to mitochondrial dysfunction and leads to colony loss/failure</a></td>
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<td><a href="/aops/200">Aop:200 - Estrogen receptor activation leading to breast cancer </a></td>
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<td><a href="/aops/273">Aop:273 - Mitochondrial complex inhibition leading to liver injury</a></td>
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<td><a href="/aops/326">Aop:326 - Thermal stress leading to population decline (3)</a></td>
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<td><a href="/aops/325">Aop:325 - Thermal stress leading to population decline (2)</a></td>
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<td><a href="/aops/324">Aop:324 - Thermal stress leading to population decline (1)</a></td>
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<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
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<td><a href="/aops/437">Aop:437 - Inhibition of mitochondrial electron transport chain (ETC) complexes leading to kidney toxicity</a></td>
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<td><a href="/aops/423">Aop:423 - Toxicological mechanisms of hepatocyte apoptosis through the PARP1 dependent cell death pathway </a></td>
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<td><a href="/aops/479">Aop:479 - Mitochondrial complexes inhibition leading to heart failure via increased myocardial oxidative stress</a></td>
<td><a href="/aops/479">Aop:479 - Mitochondrial complexes inhibition leading to left ventricular function decrease via increased myocardial oxidative stress</a></td>
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<td><a href="/aops/480">Aop:480 - Mitochondrial complexes inhibition leading to heart failure via decreased ATP production</a></td>
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<td><a href="/aops/481">Aop:481 - AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
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<td><a href="/aops/509">Aop:509 - Nrf2 inhibition leading to vascular disrupting effects through activating apoptosis signal pathway and mitochondrial dysfunction</a></td>
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<td><a href="/aops/511">Aop:511 - The AOP framework on ROS-mediated oxidative stress induced vascular disrupting effects </a></td>
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<td><a href="/aops/256">Aop:256 - Inhibition of mitochondrial DNA polymerase gamma leading to kidney toxicity</a></td>
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<td><a href="/aops/258">Aop:258 - Renal protein alkylation leading to kidney toxicity</a></td>
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<td><a href="/aops/464">Aop:464 - Calcium overload in dopaminergic neurons of the substantia nigra leading to parkinsonian motor deficits</a></td>
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<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
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<td><a href="/aops/205">Aop:205 - AOP from chemical insult to cell death</a></td>
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<td><a href="/aops/335">Aop:335 - AOP for urothelial carcinogenesis due to chemical cytotoxicity by mitochondrial impairment </a></td>
<td><a href="/aops/130">Aop:130 - Phospholipase A2 (LPLA2) inhibitors leading to hepatotoxicity</a></td>
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<td><a href="/aops/497">Aop:497 - ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
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<td><a href="/aops/34">Aop:34 - LXR activation leading to hepatic steatosis</a></td>
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<td><a href="/aops/447">Aop:447 - Kidney failure induced by inhibition of mitochondrial electron transfer chain through apoptosis, inflammation and oxidative stress pathways</a></td>
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<td><a href="/aops/207">Aop:207 - NADPH oxidase and P38 MAPK activation leading to reproductive failure in Caenorhabditis elegans</a></td>
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<td><a href="/aops/476">Aop:476 - Adverse Outcome Pathways diagram related to PBDEs associated male reproductive toxicity</a></td>
<td><a href="/aops/622">Aop:622 - Calcineurin inhibitor induced nephrotoxicity leading to kidney failure</a></td>
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<td><a href="/aops/624">Aop:624 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated mitochondrial dysfunction</a></td>
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<td><a href="/aops/627">Aop:627 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated mitochondrial dysfunction</a></td>
<p><p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Shaki et al. (2012) found that uranyl acetate (UA) exposure in isolated rat kidney mitochondria decreased the ATP production levels and ATP/ADP ratio in a concentration-dependent manner, through inhibition of complexes II and III of the ETC. Both of these levels were significantly changed at UA concentrations of 100 µM and 200 µM. In addition, a concentration-dependent decrease in activity of complex II with exposure to uranium (U</span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">) was observed (Shaki et al., 2012). They also found that mitochondrial membrane potential damage and mitochondrial swelling significantly increased both time- and dose-dependently in the treated rat kidneys (Shaki et al., 2012). ATP/ADP ratios were also decreased significantly by treatment with 100 µM or more of uranium (Shaki et al., 2012). Mitochondrial outer membrane damage was significantly decreased by treatment with 200 µM of uranium (Shaki et al., 2012).</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Shaki et al. (2013) also investigated the effects of uranium on rat kidneys. They found that mitochondrial permeability transition was also impacted by uranium treatment, causing increased mitochondrial swelling and increased disruption of energy homeostasis (Shaki et al., 2013). </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Hao et al. (2014) assessed the changes in mitochondrial potential in human kidney proximal tubular cells treated with uranium and found that the group treated with 500 µM of depleted uranium for 24 hours showed a significant decrease in mitochondrial membrane potential.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">In their study of the effects of depleted uranium treatment on human embryonic kidney cells, Hao et al. (2016) found that ETHE1, a mitochondrial protein involved in mitochondrial homeostasis and mitochondrial diseases, had significant dose- and time-dependant decreases in gene expression when treated with 125 µM or more depleted uranium (DU)</span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"> for 2 hours or more. </span></span></span></span></p>
</p>
<h4>Nanoparticles and Micrometer Particles</h4>
<p><p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Karlsson et al. (2009) conducted experiments to examine the effects of micrometer and nanoparticle treatments of copper and iron on human alveolar type-II epithelial cells. Their results showed that copper oxide micrometer and nanoparticle treatments were able to cause dose-dependant mitochondrial depolarization with doses as low as 5 µg/cm<sup>2</sup> (Karlsson et al., 2009). Iron(III) oxide nanoparticles and micrometer particles were both able to cause similar amounts of mitochondrial depolarization, along with iron (IV) oxide micrometer particles, however they were all much less toxic than copper oxide nanoparticles or micrometer particles (Karlsson et al., 2009).</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">The effects of gold nanoparticle (Au1.4MS) treatment on human cervical cancer cells were assessed by Pan et al. (2009), who found that the treated cells experienced a significant increase in permeability transition. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-family:"Times New Roman",serif; font-size:12pt">Huerta-García et al. (2014) studied the effects of titanium oxide nanoparticle treatment on glial tumor rat neuronal cells and cancerous human brain cells. Their results showed that in the treated rat and human cells there was a clear time-dependant increase in depolarization (Huerta-García et al., 2014). Both the human and rat cells showed time-dependant decreases in mitochondrial membrane potential. The TiO<sub>2</sub> nanoparticles were more toxic</span><span style="font-family:Calibri, sans-serif"><span style="font-size:10.6667px"> </span></span><span style="font-family:Calibri,sans-serif; font-size:12pt"><span style="font-family:"Times New Roman",serif">to the human cells than to the rat cells. The human cells showed a significant decrease</span></span><span style="font-family:Calibri,sans-serif; font-size:12pt"><span style="font-family:"Times New Roman",serif"> in mitochondrial membrane potential as early as 2 hours post-treatment, while the rat cells did not show significant decrease until 6 hours post-treatment (Huerta-García et al., 2014).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Zhang et al. (2018) investigated the effects of copper nanoparticles on mitochondrial membrane potential in pig kidney cells and found that the treated cells showed a dose-dependant increase in the rate of mitochondrial membrane potential change from 40 µg/mL to 80 µg/mL when treated for 12 hours. </span></span></span></span></p>
</p>
<h4>Cadmium</h4>
<p><p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Belyaeva et al. (2012) studied the effects of cadmium treatment on rat kidney cells. In particular, they looked at different respiration rates in treated and untreated rat kidney cell lines. They found that resting respiration rates were significantly stimulated at 48 hours of treatment with 100 µM of cadmium, while uncoupled respiration was unaffected, and basal respiration was enhanced (Belyaeva et al., 2012). These changes in respiration imply that cadmium was capable of reducing the uncoupling efficiency of the cells at concentrations of 100 µM or higher. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Miccadei and Floridi (1993) studied changes in oxygen consumption in rat liver mitochondria which had been treated with cadmium. Their results showed that the treated rats showed a dose-dependant decrease in oxygen consumption which began with doses as low as 3 µM (Miccadei and Floridi, 1993). </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Wang et al. (2009), found that, when applied together, lead and cadmium showed individual inhibition and additive effects of rat kidney mitochondrial COX gene expression. </span></span></span></span></p>
<p>Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (drosophila, C, elegans) are considered as potential models to study mitochondrial function. New data on marine invertebrates, such as molluscs and crustaceans and non-Drosophila species, are emerging (Martinez-Cruz et al., 2012). Mitochondrial dysfunction can be measured in animal models used for toxicity testing (Winklhofer and Haass, 2010; Waerzeggers et al., 2010) as well as in humans (Winklhofer and Haass, 2010).</p>
<p><strong>- Revision of AOP3 (Project: </strong><a href="https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms" rel="noreferrer noopener" target="_blank">NP/EFSA/PREV/2024/02</a><strong>)</strong>: Endogenous ROS formation by complex I: In mammals, complex I is a dominant site of mitochondrial ROS, especially via RET. In plants (Senkler et al. 2017; Maldonado), mitochondria contain alternative NAD(P)H dehydrogenases and an alternative oxidase (AOX) that bypass Complex I and III These pathways reduce ROS formation by preventing over-reduction of the ETC. Complex I still produces ROS, but generally less damaging due to AOX. Yeast: S. cerevisiae lacks a canonical Complex I entirely, relying instead on alternative NADH dehydrogenases. Consequently, mitochondrial ROS production from a Complex I-like source is absent. Other fungi with true Complex I (e.g., Neurospora crassa) do generate ROS similar to animals. <strong>- Not endorsed</strong></p>
<h4>Key Event Description</h4>
<p>Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress.</p>
<p>Mitochondria can be found in all cells and are considered the most important cellular consumers of oxygen. Furthermore, mitochondria possess numerous redox enzymes capable of transferring single electrons to oxygen, generating the superoxide (O2-). Some mitochondrial enzymes that are involved in reactive oxygen species (ROS) generation include the electron-transport chain (ETC) complexes I, II and III; pyruvate dehydrogenase (PDH) and glycerol-3-phosphate dehydrogenase (GPDH). The transfer of electrons to oxygen, generating superoxide, happens mainly when these redox carriers are charged enough with electrons and the potential energy for transfer is elevated, like in the case of high mitochondrial membrane potential. In contrast, ROS generation is decreased if there are not enough electrons and the potential energy for the transfer is not sufficient (reviewed in Lin and Beal, 2006).</p>
<p>Cells are also able to detoxify the generated ROS due to an extensive antioxidant defence system that includes superoxide dismutases, glutathione peroxidases, catalase, thioredoxins, and peroxiredoxins in various cell organelles (reviewed in Lin and Beal, 2006). It is worth mentioning that, as in the case of ROS generation, antioxidant defences are also closely related to the redox and energetic status of mitochondria. If mitochondria are structurally and functionally healthy, an antioxidant defence mechanism balances ROS generation, and there is not much available ROS production. However, in case of mitochondrial damage, the antioxidant defence capacity drops and ROS generation takes over. Once this happens, a vicious cycle starts and ROS can further damage mitochondria, leading to more free-radical generation and further loss of antioxidant capacity. During mitochondrial dysfunction the availability of ATP also decreases, which is considered necessary for repair mechanisms after ROS generation.</p>
<p>A number of proteins bound to the mitochondria or endoplasmic reticulum (ER), especially in the mitochondria-associated ER membrane (MAM), are playing an important role of communicators between these two organelles (reviewed Mei et al., 2013). ER stress induces mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production (reviewed Mei et al., 2013). Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis. At the same, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family. ER stress activates ERO1 and leads to excessive production of ROS, which, in turn, inactivates SERCA and activates inositol-1,4,5- trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction. Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress (reviewed Mei et al., 2013). For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress. Increased Ca2+ flux triggers loss of mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (mPTP), release of cytochrome c and apoptosis inducing factor (AIF), decreasing ATP synthesis and rendering the cells more vulnerable to both apoptosis and necrosis (Wang and Qin, 2010).</p>
Mitochondria are an important site of Ca2+ regulation and storage, taking up Ca2+ ions electrophoretically from the cytosol through a Ca2+ uniporter, which can then accumulate in the mitochondria (Roos et al., 2012; Orrenius et al., 2015). Similarities between calcium and metals, such as cadmium and lead, makes the entrance and accumulation of these metals into the mitochondria via calcium metals possible by mode of molecular mimicry (Mathews et al., 2013; Adiele et al., 2012). The outer mitochondrial membrane also contains the divalent metal transporter (DMT1), which allows for mitochondrial uptake of divalent metals such as Fe and Mn. When cells are under heavy metal-induced stress, DMT has been shown to be overexpressed in the mitochondrial membrane, making the mitochondria targets of metal toxicity and accumulation.</p>
<p>Heavy metal exposure in aerobic organisms increases ROS formation through redox cycling, where metals with different valence states (Fe, Cu, Cr, etc.) directly produce ROS as they are reduced by cellular antioxidants and then react with oxygen (Shaki et al., 2012; Shaki et al., 2013; Pourahmad et al., 2006; Santos et al., 2007). The production of highly reactive hydroxyl radicals under mitochondrial oxidative stress and in the presence of transition metals occurs via the Fenton reaction or Haber-Weiss reaction (Hancock et al., 2001; Valko et al., 2005; Adam-Vizi et al., 2010). Metals and ROS are capable of damaging mitochondrial DNA as well as mechanisms of DNA repair and proliferation arrest (Valko et al., 2005). Metals and ROS have the potential to directly damage mitochondrial membranes and structure by binding to and oxidizing membrane lipids and proteins. This structural damage can collapse the MMP and lead to the opening of the MPTP (Orrenius et al., 2015; Roos et al., 2012; Pourahmad et al., 2006). Uranium and mercury, for example, have both been shown to directly inhibit the mitochondrial electron transport chain and interfere with ATP production (Shaki et al., 2012; Roos et al., 2012). Furthermore, as previously mentioned, metals have been shown to inhibit ROS-detoxifying enzymes. By binding to these enzymes, metals can inhibit their antioxidant functions, and cause an accumulation of ROS and increased synthesis of more antioxidant enzymes in order to combat the oxidative stress (Blajszczak and Bonini, 2017).</p>
<p><strong>Summing up:</strong> Mitochondria play a pivotal role in cell survival and cell death because they are regulators of both energy metabolism and apoptotic/necrotic pathways (Fiskum, 2000; Wieloch, 2001; Friberg and Wieloch, 2002). The production of ATP via oxidative phosphorylation is a vital mitochondrial function (Kann and Kovács, 2007; Nunnari and Suomalainen, 2012). The ATP is continuously required for signalling processes (e.g. Ca2+ signalling), maintenance of ionic gradients across membranes, and biosynthetic processes (e.g. protein synthesis, heme synthesis or lipid and phospholipid metabolism) (Kang and Pervaiz, 2012), and (Green, 1998; McBride et al., 2006). Inhibition of mitochondrial respiration contributes to various cellular stress responses, such as deregulation of cellular Ca2+ homeostasis (Graier et al., 2007) and ROS production (Nunnari and Suomalainen, 2012; reviewed Mei et al., 2013).). It is well established in the existing literature that mitochondrial dysfunction may result in: (a) an increased ROS production and a decreased ATP level, (b) the loss of mitochondrial protein import and protein biosynthesis, (c) the reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, (d) the loss of the mitochondrial membrane potential, (e) the loss of mitochondrial motility, causing a failure to re-localize to the sites with increased energy demands (f) the destruction of the mitochondrial network, and (g) increased mitochondrial Ca2+ uptake, causing Ca2+ overload (reviewed in Lin and Beal, 2006; Graier et al., 2007), (h) the rupture of the mitochondrial inner and outer membranes, leading to (i) the release of mitochondrial pro-death factors, including cytochrome c (Cyt. c), apoptosis-inducing factor, or endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013), which eventually leads to apoptotic, necrotic or autophagic cell death (Wang and Qin, 2010). Due to their structural and functional complexity, mitochondria present multiple targets for various compounds.</p>
<h4>How it is Measured or Detected</h4>
<p>Mitochondrial dysfunction can be detected using isolated mitochondria, intact cells or cells in culture as well as in vivo studies. Such assessment can be performed with a large range of methods (revised by Brand and Nicholls, 2011) for which some important examples are given. All approaches to assess mitochondrial dysfunction fall into two main categories: the first assesses the consequences of a loss-of-function, i.e. impaired functioning of the respiratory chain and processes linked to it. Some assay to assess this have been described for KE1, with the limitation that they are not specific for complex I. In the context of overall mitochondrial dysfunction, the same assays provide useful information, when performed under slightly different assay conditions (e.g. without addition of complex III and IV inhibitors). The second approach assesses a ‘non-desirable gain-of-function’, i.e. processes that are usually only present to a very small degree in healthy cells, and that are triggered in a cell, in which mitochondria fail.</p>
<p>I. Mitochondrial dysfunction assays assessing a loss-of function.</p>
<p>1. Cellular oxygen consumption.</p>
<p>See KE1 for details of oxygen consumption assays. The oxygen consumption parameter can be combined with other endpoints to derive more specific information on the efficacy of mitochondrial function. One approach measures the ADP-to-O ratio (the number of ADP molecules phosphorylated per oxygen atom reduced (Hinkle, 1995 and Hafner et al., 1990). The related P/O ratio is calculated from the amount of ADP added, divided by the amount of O<sub>2</sub> consumed while phosphorylating the added ADP (Ciapaite et al., 2005; Diepart et al., 2010; Hynes et al., 2006; James et al., 1995; von Heimburg et al., 2005).</p>
<p>The mitochondrial membrane potential (Δψm) is the electric potential difference across the inner mitochondrial membrane. It requires a functioning respiratory chain in the absence of mechanisms that dissipate the proton gradient without coupling it to ATP production. The classical, and still most quantitative method uses a tetraphenylphosphonium ion (TPP+)-sensitive electrode on suspensions of isolated mitochondria. The Δψm can also be measured in live cells by fluorimetric methods. These are based on dyes which accumulate in mitochochondria because of Δψm. Frequently used are tetramethylrhodamineethylester (TMRE), tetramethylrhodaminemethyl ester (TMRM) (Petronilli et al., 1999) or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide (JC-1). Mitochondria with intact membrane potential concentrate JC-1, so that it forms red fluorescent aggregates, whereas de-energized mitochondria cannot concentrate JC-1 and the dilute dye fluoresces green (Barrientos et al., 1999). Assays using TMRE or TMRM measure only at one wavelength (red fluorescence), and depending on the assay setup, de-energized mitochondria become either less fluorescent (loss of the dye) or more fluorescent (attenuated dye quenching).</p>
<p><strong>- Revision of AOP3 (Project: </strong><a href="https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms" rel="noreferrer noopener" target="_blank">NP/EFSA/PREV/2024/02</a><strong>):</strong> The mitochondrial membrane potential (Δψm) is the electric potential difference across the inner mitochondrial membrane. It requires a functioning respiratory chain in the absence of mechanisms that dissipate the proton gradient without coupling it to ATP production. Quantitative assessment of ΔΨm in living cells is most commonly achieved through the use of cationic, lipophilic fluorescent probes that accumulate within the mitochondrial matrix in proportion to the electrochemical gradient (Leonard et al., 2014). Among these, tetramethylrhodamine derivatives such as TMRE (tetramethylrhodamineethyl ester) and TMRM (tetramethylrhodamine methyl ester) are widely employed due to their reversible, potential-dependent distribution across the inner mitochondrial membrane (Scaduto and Grotyohann, 1999; Creed and McKenzie, 2019). When applied at non-quenching, nanomolar concentrations, these dyes allow linear and quantitative detection of ΔΨm, as fluorescence intensity directly correlates with mitochondrial polarization. Detection can be performed by flow cytometry for population-level quantification, by high-content microscopy for spatially resolved analysis, or by fluorescence plate readers for higher throughput (Wong and Cortopassi, 2002; Valdebenito and Dunchen, 2022). Quantitative interpretation requires the use of appropriate controls, typically involving treatment with protonophores such as FCCP or CCCP, which fully dissipate ΔΨm and thereby establish baseline fluorescence, and inhibitors such as oligomycin or antimycin A to reveal different components of mitochondrial respiration. In parallel, dyes such as JC-1 are also used, though their ratiometric readout is less sensitive at low potentials and more prone to artifacts compared with TMRE or TMRM (Leonard et al., 2022). For accurate normalization, measurements are often corrected for cell number, mitochondrial content, or total protein, and fluorescence changes are expressed relative to maximal depolarization. In addition to chemical probes, genetically encoded sensors, such as mitochondria-targeted fluorescent proteins fused to potential-sensitive domains, provide complementary tools for ΔΨm monitoring in live-cell and in vivo contexts (Leonard et al., 2022). <strong>- Not endorsed</strong> </p>
<p>3. Enzymatic activity of the electron transport system (ETS).</p>
<p>Determination of ETS activity can be dene following Owens and King's assay (1975). The technique is based on a cell-free homogenate that is incubated with NADH to saturate the mitochondrial ETS and an artificial electron acceptor [l - (4 -iodophenyl) -3 - (4 -nitrophenyl) -5-phenylte trazolium chloride (INT)] to register the electron transmission rate. The oxygen consumption rate is calculated from the molar production rate of INT-formazan which is determined spectrophotometrically (Cammen et al., 1990).</p>
<p>4. ATP content.</p>
<p>For the evaluation of ATP levels, various commercially-available ATP assay kits are offered based on luciferin and luciferase activity. For isolated mitochondria various methods are available to continuously measure ATP with electrodes (Laudet 2005), with luminometric methods, or for obtaining more information on different nucleotide phosphate pools (e.g. Ciapaite et al., (2005).</p>
<p><strong>Determination of mitochondrial ATP production based on extracellular flux analysis </strong></p>
<p>The method is based on the detection of OCR (Oxygen Consumption Rate) that represents mitochondrial respiration as well as on the detection of ECAR (extracellular acidification rate) / proton efflux rate (PER): reflects extracellular acidification, a proxy for glycolysis (lactate release) plus contributions from CO₂/HCO₃⁻. PER is preferred over raw ECAR since it corrects for CO₂-derived acidification (Desousa et al., 2023; Espinosa et al., 2022). Application of inhibitors of individual complexes of the respiratory chain allows the detection of ATP-linked OCR: portion of oxygen consumption directly driving ATP synthesis (lost after ATP synthase inhibition) (Yoo et al., 2024). The proton leak & non-mitochondrial OCR represents remaining oxygen consumption after ATP synthase and electron transport chain inhibitor addition. The difference yields the ATP-coupled respiration component. </p>
<p><strong>Calculation of mitochondrial ATP production </strong></p>
<p>Mito ATP production rate (pmol ATP/min) = OCRATP (pmol O2/min) × 2 × P/O </p>
<p>OCR_ATP: ATP-coupled portion of OCR. </p>
<p>Factor 2: each O₂ molecule contains two oxygen atoms. </p>
<p>P/O ratio: number of ATP molecules synthesized per oxygen atom reduced. A mean P/O ≈ 2.75 is typically assumed (validated across many cell types but substrate- and condition-dependent) (Plitzko and Loesgen, 2018; Mookerjee et al., 2017; Motawe et al., 2024). </p>
<p><strong>Limitations</strong> </p>
<p>P/O ratio varies by substrate (glucose vs. fatty acids), cell type, and conditions. Fixed values are approximations. </p>
<p>Non-mitochondrial oxygen consumption (oxidases, peroxidases, etc.) can confound OCR, hence use of ETC inhibitors. </p>
<p>PER vs. ECAR: CO₂-driven acidification must be corrected to avoid overestimating glycolytic ATP. </p>
<p>Normalization: results are usually expressed per cell, protein content, DNA, or mitochondrial mass — interpretation depends on normalization method. </p>
<p><span style="font-size:12.0pt"><span style="font-family:Arial"><span style="color:#212529"><span style="background-color:white"><strong>- Not endorsed</strong></span></span></span></span></p>
</div>
<p><br />
II. Mitochondrial dysfunction assays assessing a gain-of function.</p>
<p>The opening of the PTP is associated with a permeabilization of mitochondrial membranes, so that different compounds and cellular constituents can change intracellular localization. This can be measured by assessment of the translocation of cytochrome c, adenylate kinase or AIF from mitochondria to the cytosol or nucleus. The translocation can be assessed biochemically in cell fractions, by imaging approaches in fixed cells or tissues or by life-cell imaging of GFP fusion proteins (Single 1998; Modjtahedi 2006). An alternative approach is to measure the accessibility of cobalt to the mitochondrial matrix in a calcein fluorescence quenching assay in live permeabilized cells (Petronilli et al., 1999).</p>
<p>2. mtDNA damage as a biomarker of mitochondrial dysfunction.</p>
<p>Various quantitative polymerase chain reaction (QPCR)-based assays have been developed to detect changes of DNA structure and sequence in the mitochondrial genome. mtDNA damage can be detected in blood after low-level rotenone exposure, and the damage persists even after CI activity has returned to normal. With a more sustained rotenone exposure, mtDNA damage is also detected in skeletal muscle. These data support the idea that mtDNA damage in peripheral tissues in the rotenone model may provide a biomarker of past or ongoing mitochondrial toxin exposure (Sanders et al., 2014a and 2014b).</p>
<p>3. Generation of ROS and resultant oxidative stress.</p>
<p>a. General approach. Electrons from the mitochondrial ETS may be transferred ‘erroneously’ to molecular oxygen to form superoxide anions. This type of side reaction can be strongly enhanced upon mitochondrial damage. As superoxide may form hydrogen peroxide, hydroxyl radicals or other reactive oxygen species, a large number of direct ROS assays and assays assessing the effects of ROS (indirect ROS assays) are available (Adam-Vizi, 2005; Fan and Li 2014). Direct assays are based on the chemical modification of fluorescent or luminescent reporters by ROS species. Indirect assays assess cellular metabolites, the concentration of which is changed in the presence of ROS (e.g. glutathione, malonaldehyde, isoprostanes,etc.) At the animal level the effects of oxidative stress are measured from biomarkers in the blood or urine.</p>
<p>b. Measurement of the cellular glutathione (GSH) status. GSH is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase (GSSH + NADPH + H+ à 2 GSH + NADP+). The ratio of GSH/GSSG is therefore a good indicator for the cellular NADH+/NADPH ratio (i.e. the redox potential). GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically with DTNB (Ellman’s reagent). As excess GSSG is rapidly exported from most cells to maintain a constant GSH/GSSG ratio, a reduction of total glutathione (GSH/GSSG) is often a good surrogate measure for oxidative stress.</p>
<p>c. Quantification of lipid peroxidation. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA)-reactive compounds such as malondialdehyde generated from the decomposition of cellular membrane lipid under oxidative stress (Pryor et al., 1976). This method is quite sensitive, but not highly specific. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be more specific for lipid peroxidation. A number of commercial ELISA kits have been developed for IsoPs, but interfering agents in samples requires partial purification before analysis. Alternatively, GC/MS may be used, as robust (specific) and sensitive method.</p>
<p><br />
d. Detection of superoxide production. Generation of superoxide by inhibition of complex I and the methods for its detection are described by Grivennikova and Vinogradov (2014). A range of different methods is also described by BioTek (<a class="external free" href="http://www.biotek.com/resources/articles/reactive-oxygen-species.html" rel="nofollow" target="_blank">http://www.biotek.com/resources/articles/reactive-oxygen-species.html</a>). The reduction of ferricytochrome c to ferrocytochrome c may be used to assess the rate of superoxide formation (McCord, 1968). Like in other superoxide assays, specificity can only be obtained by measurements in the absence and presence of superoxide dismutase. Chemiluminescent reactions have been used for their increased sensitivity. The most widely used chemiluminescent substrate is lucigenin. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical, and they become membrane impermeable after oxidation (trapping at site of formation). The best characterized of these probes are Hydro-Cy3 and Hydro-Cy5. generation of superoxide in mitochondria can be visualized using fluorescence microscopy with MitoSOX™ Red reagent (Life Technologies). MitoSOX™ Red reagent is a cationic derivative of dihydroethidium that permeates live cells and accumulates in mitochondria.</p>
<p>d. Detection of superoxide production. Generation of superoxide by inhibition of complex I and the methods for its detection are described by Grivennikova and Vinogradov (2014). A range of different methods is also described by BioTek (<a class="external free" href="http://www.biotek.com/resources/articles/reactive-oxygen-species.html" rel="nofollow" target="_blank">http://www.biotek.com/resources/articles/reactive-oxygen-species.html</a>). The reduction of ferricytochrome c to ferrocytochrome c may be used to assess the rate of superoxide formation (McCord, 1968). Like in other superoxide assays, specificity can only be obtained by measurements in the absence and presence of superoxide dismutase. Chemiluminescent reactions have been used for their increased sensitivity. The most widely used chemiluminescent substrate is lucigenin. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical, and they become membrane impermeable after oxidation (trapping at site of formation). The best characterized of these probes are Hydro-Cy3 and Hydro-Cy5. generation of superoxide in mitochondria can be visualized using fluorescence microscopy with MitoSOX™ Red reagent (Life Technologies). MitoSOX™ Red reagent is a cationic derivative of dihydroethidium that permeates live cells and accumulates in mitochondria.</p>
<p>e. Detection of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) production. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products in the presence of hydrogen peroxide (Zhou et al., 1997: Ruch et al., 1983). The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H<sub>2</sub>O<sub>2</sub> form increasing amounts of fluorescent product (Tarpley et al., 2004).</p>
<p>Summing up, mitochondrial dysfunction can be measured by: • ROS production: superoxide (O2-), and hydroxyl radicals (OH−) • Nitrosative radical formation such as ONOO− or directly by: • Loss of mitochondrial membrane potential (MMP) • Opening of mitochondrial permeability transition pores (mPTP) • ATP synthesis • Increase in mitochondrial Ca2+ • Cytochrome c release • AIF (apoptosis inducing factor) release from mitochondria • Mitochondrial Complexes enzyme activity • Measurements of mitochondrial oxygen consumption • Ultrastructure of mitochondria using electron microscope and mitochondrial fragmentation measured by labelling with DsRed-Mito expression (Knott et al, 2008) Mitochondrial dysfunction-induced oxidative stress can be measured by: • Reactive carbonyls formations (proteins oxidation) • Increased 8-oxo-dG immunoreactivity (DNA oxidation) • Lipid peroxidation (formation of malondialdehyde (MDA) and 4- hydroxynonenal (HNE) • 3-nitrotyrosine (3-NT) formation, marker of protein nitration • Translocation of Bid and Bax to mitochondria • Measurement of intracellular free calcium concentration ([Ca2+]i): Cells are loaded with 4 μM fura-2/AM). • Ratio between reduced and oxidized form of glutathione (GSH depletion) (Promega assay, TB369; Radkowsky et al., 1986) • Neuronal nitric oxide synthase (nNOS) activation that is Ca2+-dependent. All above measurements can be performed as the assays for each readout are well established in the existing literature (e.g. Bal-Price and Brown, 2000; Bal-Price et al., 2002; Fujikawa, 2015; Walker et al., 1995). See also KE <a href="/wiki/index.php/Event:209" title="Event:209"> Oxidative Stress, Increase</a></p>
<p><strong>Assay Type & Measured Content</strong></p>
</td>
<td><strong>Description</strong></td>
<td><strong>Dose Range Studied</strong></td>
<td>
<p><strong>Assay Characteristics</strong></p>
<p><strong>(Length/Ease of use/Accuracy)</strong></p>
</td>
</tr>
<tr>
<td>
<p><strong>Rhodamine 123 Assay</strong></p>
<p>Measuring Mitochondrial membrane potential (MMP) and its collapse </p>
<p>(Shaki et al., 2012)</p>
</td>
<td>
<p>Mitochondrial uptake of cationic fluorescent dye, rhodamine 123, is used for estimation of mitochondrial membrane potential. The fluorescence was monitored using Schimadzou RF-5000U fluorescence spectrophotometer at the excitation and emission wavelength of 490 nm and 535 nm, respectively.</p>
<td>Laser scanning confocal microscopy in combination with the potentiometric fluorescence dye tetramethylrhodamine ethyl ester to monitor relative changes in membrane potential in single isolated cardiac mitochondria. The cationic dye distributes across the membrane in a voltage-dependent manner. Therefore, the large potential gradient across the inner mitochondrial membrane results in the accumulation of the fluorescent dye within the matrix compartment. Rapid depolarizations are caused by the opening of the transition pore.</td>
<p>Measuring cellular glutathione (GSH) status; ratio of GSH/GSSG</p>
<p>(Owen & Butterfield, 2010; Shaki et al., 2013)</p>
</td>
<td>GSH and GSSG levels are determinted biochemically with DTNB (Ellman’s reagent). The developed yellow color was read at 412 nm on a spectrophotometer.</td>
<td>100 µM uranyl acetate</td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>TBARS Assay</strong></p>
<p>Quantification of lipid peroxidation</p>
<p>(Yuan et al., 2016)</p>
</td>
<td>MDA content, a product of lipid peroxidation, was measured using a thiobarbituric acid reactive substances (TBARS) assay. Briefly, the kidney cells were collected in 1 ml PBS buffer solution (pH 7.4) and sonicated. MDA reacts with thiobarbituric acid forming a colored product which can be measured at an absorbance of 532 nm.</td>
<p>Increase in mitochondrial Ca<sup>2+</sup> influx</p>
<p>(Pozzan & Rudolf, 2009)</p>
</td>
<td>Together with GFP, the aequorin moiety acts as Ca<sup>2+</sup> sensor <em>in vivo</em>, which delivers emission energy to the GFP acceptor molecule in a BRET (Bioluminescence Resonance Energy Transfer) process; the Ca2+ can then be visualized with fluorescence microscopy.</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Western blot & immunostaining analyses</strong></p>
<p>Measuring cytochrome c release</p>
(Chen et al., 2000)</td>
<td>Examining the redistribution of Cyto c in cytosolic and mitochondrial cellular fractions. Cells are homogenized and centrifuged, then prepared for immunoblots. Cellular fractions were washed in PBS and lysed in 1% NP-40 buffer. Cellular proteins were separated by SDS–PAGE, transferred onto nitrocellulose membranes, probed using immunoblot analyses with antibodies specific to cyto c (6581A for Western and 65971A for immunostaining; Pharmingen)</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Quantikine Rat/Mouse Cytochrome c Immunoassay</strong></p>
<p>Measuring cytochrome c release</p>
<p>(Shaki et al., 2012)</p>
</td>
<td>Cytochrome C release was measured a monoclonal antibody specific for rat/mouse cytochrome c was precoated onto the microplate. Seventy-five microliter of conjugate (containing mono- clonal antibody specific for cytochrome c conjugated to horseradish peroxidase). After 2 h of incubation, the substrate solution (100 μl) was added to each well and incubated for 30 min. After 100 μl of the stop solution was added to each well; the optical density of each well was determined by the aforementioned microplate spectrophotometer set to 450 nm.</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Membrane potential and cell viability – Flow Cytometry</strong></p>
<p>Measuring cytochrome c release</p>
<p>(Kruidering et al., 1997)</p>
</td>
<td>“Dc and viability were determined by analyzing the R123 and propidium iodide fluorescence intensity with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser, with the Lysis software program (Becton Dickinson). R123 is a cationic dye that accumulates in the negatively charged inner side of the mitochondria. When the potential drops, less R123 accumulates in the mitochondria, which results in a lower fluorescence signal. The potential was measured as follows: at the indicated times, a 500-ml sample of the cell suspension was taken and transferred to an Eppendorf minivial. To this sample, 100 ml of 6 mM R123 in buffer D was added. After incubation for 10 min at 37°C, the cell suspension was centrifuged for 5 min at 80 3 <em>g</em>. The cell pellet was resuspended in 200 ml of buffer D, containing 0.2 mM R123 and 10 mM propidium iodide, to prevent loss of R123 and to stain nonviable cells, respectively. The samples were transferred to FACScan tubes and analyzed immediately. Analysis was performed at a flow rate of<br />
60 ml/min. R123 fluorescence was detected by the FL1 detector with an emission detection limit below 560 nm. Propidium iodide fluorescence was detected by the FL3 detector, with emission detection above 620 nm. Per sample 3,000 to 5,000 cells were counted (Van de Water <em>et al.</em>, 1993)”</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<h4>References</h4>
<p> </p>
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<p>Adam-Vizi, V., & Starkov, A. A. (2010). Calcium and mitochondrial reactive oxygen species generation: How to read the facts. Journal of Alzheimer's Disease : JAD, 20 Suppl 2, S413-S426. doi:10.3233/JAD-2010-100465</p>
<p>Adam-Vizi V. Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxid Redox Signal. 2005, 7(9-10):1140-1149.</p>
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<p>Bal-Price A. and Guy C. Brown. Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria. J. Neurochemistry, 2000, 75: 1455-1464.</p>
<p>Bal-Price A, Matthias A, Brown GC., Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production. J. Neurochem. 2002, 80: 73-80.</p>
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<p>Hynes, J.. Marroquin, L.D Ogurtsov, V.I. Christiansen, K.N. Stevens, G.J. Papkovsky, D.B. Will, Y. (2006)) Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes, Toxicol. Sci. 92 186–200.</p>
<p>James, P.E. Jackson, S.K.. Grinberg, O.Y Swartz, H.M. (1995) The effects of endotoxin on oxygen consumption of various cell types in vitro: an EPR oximetry study, Free Radic. Biol. Med. 18 (1995) 641–647.</p>
<p>Kang J, Pervaiz S. (2012). Mitochondria: Redox Metabolism and Dysfunction. Biochem Res Int 2012:896751.</p>
<p>Kann O, Kovács R. (2007). Mitochondria and neuronal activity. Am J Physiol Cell Physiol 292:C641-576.</p>
<p>Karlsson, H. L., Gustafsson, J., Cronholm, P., & Möller, L. (2009). Size-dependent toxicity of metal oxide particles—A comparison between nano- and micrometer size. Toxicology Letters, 188(2), 112-118. doi:<a href="https://doi.org/10.1016/j.toxlet.2009.03.014" target="_blank">10.1016/j.toxlet.2009.03.014</a></p>
<p>Knott Andrew B., Guy Perkins, Robert Schwarzenbacher & Ella Bossy-Wetzel. Mitochondrial fragmentation in neurodegeneration. Nature Reviews Neuroscience, 2008, 229: 505-518.</p>
<p>Kruidering, M., Van De Water, B., De Heer, E., Mulder, G. J., & Nagelkerke, J. F. (1997). Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: Mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain. The Journal of Pharmacology and Experimental Therapeutics, 280(2), 638-649.</p>
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<p>Leonard AP, Cameron RB, Speiser JL, Wolf BJ, Peterson YK, Schnellmann RG, Beeson CC, Rohrer B. Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning. Biochim Biophys Acta. 2015 Feb;1853(2):348-60. doi: 10.1016/j.bbamcr.2014.11.002. Epub 2014 Nov 13. PMID: 25447550; PMCID: PMC4289477. </p>
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<td><a href="/aops/383">Aop:383 - Inhibition of Angiotensin-converting enzyme 2 leading to liver fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/382">Aop:382 - Angiotensin II type 1 receptor (AT1R) agonism leading to lung fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/384">Aop:384 - Hyperactivation of ACE/Ang-II/AT1R axis leading to chronic kidney disease </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/409">Aop:409 - Frustrated phagocytosis leads to malignant mesothelioma</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/413">Aop:413 - Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/416">Aop:416 - Aryl hydrocarbon receptor activation leading to lung cancer through IL-6 toxicity pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/418">Aop:418 - Aryl hydrocarbon receptor activation leading to impaired lung function through AHR-ARNT toxicity pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/386">Aop:386 - Deposition of ionizing energy leading to population decline via inhibition of photosynthesis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/387">Aop:387 - Deposition of ionising energy leading to population decline via mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/319">Aop:319 - Binding to ACE2 leading to lung fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/451">Aop:451 - Interaction with lung resident cell membrane components leads to lung cancer</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/476">Aop:476 - Adverse Outcome Pathways diagram related to PBDEs associated male reproductive toxicity</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/492">Aop:492 - Glutathione conjugation leading to reproductive dysfunction via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/497">Aop:497 - ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/505">Aop:505 - Reactive Oxygen Species (ROS) formation leads to cancer via inflammation pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/513">Aop:513 - Reactive Oxygen (ROS) formation leads to cancer via Peroxisome proliferation-activated receptor (PPAR) pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/521">Aop:521 - Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/462">Aop:462 - Activation of reactive oxygen species leading the atherosclerosis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/299">Aop:299 - Deposition of energy leading to population decline via DNA oxidation and follicular atresia</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/311">Aop:311 - Deposition of energy leading to population decline via DNA oxidation and oocyte apoptosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/327">Aop:327 - Excessive reactive oxygen species production leading to mortality (1)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/328">Aop:328 - Excessive reactive oxygen species production leading to mortality (2)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/329">Aop:329 - Excessive reactive oxygen species production leading to mortality (3)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/330">Aop:330 - Excessive reactive oxygen species production leading to mortality (4)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/26">Aop:26 - Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/534">Aop:534 - Succinate dehydrogenase (SDH) inhibition leads to cancer through oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/273">Aop:273 - Mitochondrial complex inhibition leading to liver injury</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/488">Aop:488 - Increased reactive oxygen species production leading to decreased cognitive function</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/298">Aop:298 - Increase in reactive oxygen species (ROS) leading to human treatment-resistant gastric cancer</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/27">Aop:27 - Cholestatic Liver Injury induced by Inhibition of the Bile Salt Export Pump (ABCB11)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/511">Aop:511 - The AOP framework on ROS-mediated oxidative stress induced vascular disrupting effects </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/207">Aop:207 - NADPH oxidase and P38 MAPK activation leading to reproductive failure in Caenorhabditis elegans</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/423">Aop:423 - Toxicological mechanisms of hepatocyte apoptosis through the PARP1 dependent cell death pathway </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/481">Aop:481 - AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/282">Aop:282 - Adverse outcome pathway on photochemical toxicity initiated by light exposure</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/569">Aop:569 - Decreased DNA methylation of FAM50B/PTCHD3 leading to IQ loss of children via PI3K-Akt pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/613">Aop:613 - Peroxisome proliferator-activated receptor alpha activation leading to early life stage mortality via increased reactive oxygen species production</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/622">Aop:622 - Calcineurin inhibitor induced nephrotoxicity leading to kidney failure</a></td>
<p>ROS is a normal constituent found in all organisms.</p>
<p>ROS is a normal constituent found in all organisms, <em>lifestages, and sexes.</em></p>
<h4>Key Event Description</h4>
<p>Biological State: increased reactive oxygen species (ROS)</p>
<p>Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.</p>
<p>Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
<p>Reactive oxygen species (ROS) are O<sub>2</sub>- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). </p>
<div>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Reactive oxygen species (ROS) refers to the chemical species superoxide, hydrogen peroxide, and their secondary reactive products. In the biological context, ROS are signaling molecules with important roles in cell energy metabolism, cell proliferation, and fate. Therefore, balancing ROS levels at the cellular and tissue level is an important part of many biological processes. Disbalance, mainly an increase in ROS levels, can cause cell dysfunction and irreversible cell damage.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are produced from both exogenous stressors and normal endogenous cellular processes, such as the mitochondrial electron transport chain (ETC). Inhibition of the ETC can result in the accumulation of ROS. Exposure to chemicals, heavy metal ions, or ionizing radiation can also result in increased production of ROS. Chemicals and heavy metal ions can deplete cellular antioxidants reducing the cell’s ability to control cellular ROS and resulting in the accumulation of ROS. Cellular antioxidants include glutathione (GSH), protein sulfhydryl groups, superoxide dismutase (SOD). </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are radicals, ions, or molecules that have a single unpaired electron in their outermost shell of electrons, which can be categorized into two groups: free oxygen radicals and non-radical ROS [Liou et al., 2010]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">highly reactive lipid- or carbohydrate-derived carbonyl compounds</span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Potential sources of ROS include NADPH oxidase, xanthine oxidase, mitochondria, nitric oxide synthase, cytochrome P450, lipoxygenase/cyclooxygenase, and monoamine oxidase [Granger et al., 2015]. ROS are generated through NADPH oxidases consisting of p47<sup>phox</sup> and p67<sup>phox</sup>. ROS are generated through xanthine oxidase activation in sepsis [Ramos et al., 2018]. Arsenic produces ROS [Zhang et al., 2011]. Mitochondria-targeted paraquat and metformin mediate ROS production [Chowdhury et al., 2020]. ROS are generated by bleomycin [Lu et al., 2010]. Radiation induces dose-dependent ROS production [Ji et al., 2019]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are generated in the course of cellular respiration, metabolism, cell signaling, and inflammation [Dickinson and Chang 2011; Egea et al. 2017]. Hydrogen peroxide is also made by the endoplasmic reticulum in the course of protein folding. Nitric oxide (NO) is produced at the highest levels by nitric oxide synthase in endothelial cells and phagocytes. NO production is one of the main mechanisms by which phagocytes kill bacteria [Wang et al., 2017]. The other species are produced by reactions with superoxide or peroxide, or by other free radicals or enzymes.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS activity is principally local. Most ROS have short half-lives, ranging from nano- to milliseconds, so diffusion is limited, while reactive nitrogen species (RNS) nitric oxide or peroxynitrite can survive long enough to diffuse across membranes [Calcerrada et al. 2011]. Consequently, local concentrations of ROS are much higher than average cellular concentrations, and signaling is typically controlled by colocalization with redox buffers [Dickinson and Chang 2011; Egea et al. 2017]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Although their existence is limited temporally and spatially, ROS interact with other ROS or with other nearby molecules to produce more ROS and participate in a feedback loop to amplify the ROS signal, which can increase RNS. Both ROS and RNS also move into neighboring cells, and ROS can increase intracellular ROS signaling in neighboring cells [Egea et al. 2017].</span></span></p>
<p>In the primary event, photoreactive chemicals are excited by the absorption of photon energy. The energy of the photoactivated chemicals transfer to oxygen and then generates the reactive oxygen species (ROS), including superoxide (O<sub>2</sub><sup>−</sup>) via type I reaction and singlet oxygen (<sup>1</sup>O<sub>2</sub>) via type II reaction, as principal intermediate species in phototoxic reaction (Foote, 1991, Onoue et al. , 2009).</p>
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<h4>How it is Measured or Detected</h4>
<p>Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies.</p>
<p>Yuan, Yan, et al., (2013) described ROS monitoring by using H<sub>2</sub>-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H<sub>2</sub>-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm.</p>
<p>Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013).</p>
<p>Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS.</p>
<p> </p>
<p>On the basis of the pathogenesis of drug-induced phototoxicity, a reactive oxygen species (ROS) assay was proposed to evaluate the phototoxic risk of chemicals. The ROS assay can monitor generation of ROS, such as singlet oxygen and superoxide, from photoirradiated chemicals, and the ROS data can be used to evaluate the photoreactivity of chemicals (Onoue et al. , 2014, Onoue et al. , 2013, Onoue and Tsuda, 2006). The ROS assay is a recommended approach by guidelines to evaluate the phototoxic risk of chemicals (ICH, 2014, PCPC, 2014).</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Many fluorescent compounds can be used to detect ROS, some of which are specific, and others are less specific. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・ROS can be detected by fluorescent probes such as <em>p</em>-methoxy-phenol derivative [Ashoka et al., 2020].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Chemiluminescence analysis can detect the superoxide, where some probes have a wider range for detecting hydroxyl radical, hydrogen peroxide, and peroxynitrite [Fuloria et al., 2021].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・ROS in the blood can be detected using superparamagnetic iron oxide nanoparticles (SPION)-based biosensor [Lee et al., 2020].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) can be detected with a colorimetric probe, which reacts with H<sub>2</sub>O<sub>2</sub> in a 1:1 stoichiometry to produce a bright pink colored product, followed by the detection with a standard colorimetric microplate reader with a filter in the 540-570 nm range.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・The levels of ROS can be quantified using multiple-step amperometry using a stainless steel counter electrode and non-leak Ag|AgCl reference node [Flaherty et al., 2017].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Singlet oxygen can be measured by monitoring the bleaching of <em>p</em>-nitrosodimethylaniline at 440 nm using a spectrophotometer with imidazole as a selective acceptor of singlet oxygen [Onoue et al., 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Alternative methods involve the detection of redox-dependent changes to cellular constituents such as proteins, DNA, lipids, or glutathione [Dickinson and Chang 2011; Wang et al. 2013; Griendling et al. 2016]. However, these methods cannot generally distinguish between the oxidative species behind the changes and cannot provide good resolution for the kinetics of oxidative activity.</span></span></p>
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<h4>References</h4>
<p>B.H. Park, S.M. Fikrig, E.M. Smithwick Infection and nitroblue tetrazolium reduction by neutrophils: a diagnostic aid Lancet, 2 (1968), pp. 532-534</p>
<p>Akai, K., et al. (2004). "Ability of ferric nitrilotriacetate complex with three pH-dependent conformations to induce lipid peroxidation." Free Radic Res. Sep;38(9):951-62. doi: 10.1080/1071576042000261945</p>
<p>Ashoka, A. H., et al. (2020). "Recent Advances in Fluorescent Probes for Detection of HOCl and HNO." ACS omega, 5(4), 1730-1742. doi:10.1021/acsomega.9b03420</p>
<p>B.H. Park, S.M. Fikrig, E.M. Smithwick Infection and nitroblue tetrazolium reduction by neutrophils: a diagnostic aid Lancet, 2 (1968), pp. 532-534</p>
<p>Bedard, Karen, and Karl-Heinz Krause. 2007. “The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology.” Physiological Reviews 87 (1): 245–313.</p>
<p>Bisht, Shilpa, Muneeb Faiq, Madhuri Tolahunase, and Rima Dada. 2017. “Oxidative Stress and Male Infertility.” Nature Reviews. Urology 14 (8): 470–85.</p>
<p>Brieger, K., S. Schiavone, F. J. Miller Jr, and K-H Krause. 2012. “Reactive Oxygen Species: From Health to Disease.” Swiss Medical Weekly 142 (August): w13659.</p>
<p>Calcerrada, P., et al. (2011). "Nitric oxide-derived oxidants with a focus on peroxynitrite: molecular targets, cellular responses and therapeutic implications." Curr Pharm Des 17(35): 3905-3932.</p>
<p>Chattopadhyay, Sukumar, et al. "Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants." Toxicology letters 136.1 (2002): 65-76.</p>
<p>Chowdhury, A. R., et al. (2020). "Mitochondria-targeted paraquat and metformin mediate ROS production to induce multiple pathways of retrograde signaling: A dose-dependent phenomenon." Redox Biol. doi: 10.1016/j.redox.2020.101606. PMID: 32604037; PMCID: PMC7327929.</p>
<p>Dickinson, B. C. and Chang C. J. (2011). "Chemistry and biology of reactive oxygen species in signaling or stress responses." Nature chemical biology 7(8): 504-511.</p>
<p>Drew, Barry, and Christiaan Leeuwenburgh. 2002. “Aging and the Role of Reactive Nitrogen Species.” Annals of the New York Academy of Sciences 959 (April): 66–81.</p>
<p>Egea, J., et al. (2017). "European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)." Redox biology 13: 94-162.</p>
<p>Flaherty, R. L., et al. (2017). "Glucocorticoids induce production of reactive oxygen species/reactive nitrogen species and DNA damage through an iNOS mediated pathway in breast cancer." Breast Cancer Research, 19(1), 1–13. https://doi.org/10.1186/s13058-017-0823-8</p>
<p>Foote CS. Definition of type I and type II photosensitized oxidation. Photochem Photobiol. 1991;54:659.</p>
<p>Fuloria, S., et al. (2021). "Comprehensive Review of Methodology to Detect Reactive Oxygen Species (ROS) in Mammalian Species and Establish Its Relationship with Antioxidants and Cancer." Antioxidants (Basel, Switzerland) 10(1) 128. doi:10.3390/antiox10010128</p>
<p>Go, Y. M. and Jones, D. P. (2013). "The redox proteome." J Biol Chem 288(37): 26512-26520.</p>
<p>Goud, Anuradha P., Pravin T. Goud, Michael P. Diamond, Bernard Gonik, and Husam M. Abu-Soud. 2008. “Reactive Oxygen Species and Oocyte Aging: Role of Superoxide, Hydrogen Peroxide, and Hypochlorous Acid.” Free Radical Biology & Medicine 44 (7): 1295–1304.</p>
<p>Granger, D. N. and Kvietys, P. R. (2015). "Reperfusion injury and reactive oxygen species: The evolution of a concept" Redox Biol. doi: 10.1016/j.redox.2015.08.020. PMID: 26484802; PMCID: PMC4625011.</p>
<p>Griendling, K. K., et al. (2016). "Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association." Circulation research 119(5): e39-75.</p>
<p>Griendling, Kathy K., Rhian M. Touyz, Jay L. Zweier, Sergey Dikalov, William Chilian, Yeong-Renn Chen, David G. Harrison, Aruni Bhatnagar, and American Heart Association Council on Basic Cardiovascular Sciences. 2016. “Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association.” Circulation Research 119 (5): e39–75.</p>
<p>ICH. ICH Guideline S10 Guidance on Photosafety Evaluation of Pharmaceuticals.: International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; 2014.</p>
<p>Itziou, A., et al. (2011). "In vivo and in vitro effects of metals in reactive oxygen species production, protein carbonylation, and DNA damage in land snails Eobania vermiculata." Archives of Environmental Contamination and Toxicology, 60(4), 697–707. https://doi.org/10.1007/s00244-010-9583-5</p>
<p>Ji, W. O., et al. "Quantitation of the ROS production in plasma and radiation treatments of biotargets." Sci Rep. 2019 Dec 27;9(1):19837. doi: 10.1038/s41598-019-56160-0. PMID: 31882663; PMCID: PMC6934759.</p>
<p>Kruk, J. and Aboul-Enein, H. Y. (2017). "Reactive Oxygen and Nitrogen Species in Carcinogenesis: Implications of Oxidative Stress on the Progression and Development of Several Cancer Types." Mini-Reviews in Medicinal Chemistry, 17:11. doi:10.2174/1389557517666170228115324</p>
<p>Lee, D. Y., et al. (2020). "PEGylated Bilirubin-coated Iron Oxide Nanoparticles as a Biosensor for Magnetic Relaxation Switching-based ROS Detection in Whole Blood." Theranostics, 10(5), 1997-2007. doi:10.7150/thno.39662</p>
<p>Li, Z., et al. (2020). "Inhibition of MiR-25 attenuates doxorubicin-induced apoptosis, reactive oxygen species production and DNA damage by targeting pten." International Journal of Medical Sciences, 17(10), 1415–1427. https://doi.org/10.7150/ijms.41980</p>
<p>Liou, G. Y. and Storz, P. "Reactive oxygen species in cancer." Free Radic Res. 2010 May;44(5):479-96. doi:10.3109/10715761003667554. PMID: 20370557; PMCID: PMC3880197.</p>
<p>Lu, Y., et al. (2010). "Phosphatidylinositol-3-kinase/akt regulates bleomycin-induced fibroblast proliferation and collagen production." American journal of respiratory cell and molecular biology, 42(4), 432–441. https://doi.org/10.1165/rcmb.2009-0002OC</p>
<p>Onoue, S., et al. (2013). "Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation." J Appl Toxicol. 33(11):1241-50. doi: 10.1002/jat.2776. Epub 2012 Jun 13. PMID: 22696462.</p>
<p>Onoue S, Hosoi K, Toda T, Takagi H, Osaki N, Matsumoto Y, et al. Intra-/inter-laboratory validation study on reactive oxygen species assay for chemical photosafety evaluation using two different solar simulators. Toxicology in vitro : an international journal published in association with BIBRA. 2014;28:515-23.</p>
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<p>Onoue S, Kawamura K, Igarashi N, Zhou Y, Fujikawa M, Yamada H, et al. Reactive oxygen species assay-based risk assessment of drug-induced phototoxicity: classification criteria and application to drug candidates. J Pharm Biomed Anal. 2008;47:967-72.</p>
<p>Onoue S, Seto Y, Gandy G, Yamada S. Drug-induced phototoxicity; an early<em> in vitro</em> identification of phototoxic potential of new drug entities in drug discovery and development. Current drug safety. 2009;4:123-36.</p>
<p>Onoue S, Tsuda Y. Analytical studies on the prediction of photosensitive/phototoxic potential of pharmaceutical substances. Pharmaceutical research. 2006;23:156-64.</p>
<p>Ozcan, Ayla, and Metin Ogun. 2015. “Biochemistry of Reactive Oxygen and Nitrogen Species.” In Basic Principles and Clinical Significance of Oxidative Stress, edited by Sivakumar Joghi Thatha Gowder. Rijeka: IntechOpen.</p>
<p>Parrish, A. R. 2010. “2.27 - Hypoxia/Ischemia Signaling.” In Comprehensive Toxicology (Second Edition), edited by Charlene A. McQueen, 529–42. Oxford: Elsevier.</p>
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<p>Ramos, M. F. P., et al. (2018). "Xanthine oxidase inhibitors and sepsis." Int J Immunopathol Pharmacol. 32:2058738418772210. doi:10.1177/2058738418772210</p>
<p>Ravanat, J. L., et al. (2014). "Radiation-mediated formation of complex damage to DNA: a chemical aspect overview." Br J Radiol 87(1035): 20130715.</p>
<p>Schutzendubel, A. and Polle, A. (2002). "Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization." Journal of Experimental Botany, 53(372), 1351–1365. https://doi.org/10.1093/jexbot/53.372.1351</p>
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<p>Sharma, Gunjan, Nishant Kumar Rana, Priya Singh, Pradeep Dubey, Daya Shankar Pandey, and Biplob Koch. 2017. “p53 Dependent Apoptosis and Cell Cycle Delay Induced by Heteroleptic Complexes in Human Cervical Cancer Cells.” Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie 88 (April): 218–31.</p>
<p>Silva, R., et al. (2019). "Light exposure during growth increases riboflavin production, reactive oxygen species accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains." FEMS Yeast Research, 19(1), 1–7. https://doi.org/10.1093/femsyr/foy114</p>
<p>Tsuchiya K, et al. (2005). "Oxygen radicals photo-induced by ferric nitrilotriacetate complex." Biochim Biophys Acta. 1725(1):111-9. doi:10.1016/j.bbagen.2005.05.001</p>
<p>Wang, J., et al. (2017). "Glucocorticoids Suppress Antimicrobial Autophagy and Nitric Oxide Production and Facilitate Mycobacterial Survival in Macrophages." Scientific reports, 7(1), 982. https://doi.org/10.1038/s41598-017-01174-9</p>
<p>Wang, X., et al. (2013). "Imaging ROS signaling in cells and animals." Journal of molecular medicine 91(8): 917-927.</p>
<p>Yen, Cheng Chien, et al. "Inorganic arsenic causes cell apoptosis in mouse cerebrum through an oxidative stress-regulated signaling pathway." Archives of toxicology 85 (2011): 565-575.</p>
<p>Yuan, Yan, et al. "Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway." PloS one 8.5 (2013): e64330.</p>
<p>Zhang, Z., et al. (2011). "Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/β-catenin pathway in human colorectal adenocarcinoma DLD1 cells. " Toxicology and Applied Pharmacology, 256(2), 114-121. doi:10.1016/j.taap.2011.07.016</p>
<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/502">Aop:502 - Decrease, cholesterol synthesis leads to orofacial clefting</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/441">Aop:441 - Ionizing radiation-induced DNA damage leads to microcephaly via apoptosis and premature cell differentiation</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/535">Aop:535 - Binding and activation of GPER leading to learning and memory impairments</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<p>・Apoptosis is induced in human prostate cancer cell lines (<em>Homo sapiens</em>) [Parajuli et al., 2014].</p>
<p>・Apoptosis occurs in B6C3F1 mouse (<em>Mus musculus</em>) [Elmore, 2007].</p>
<p>・Apoptosis occurs in Sprague-Dawley rat (<em>Rattus norvegicus</em>) [Elmore, 2007].</p>
<p>・Apoptosis occurs in the nematode (<em>Caenorhabditis elegans</em>) [Elmore, 2007].</p>
<ul>
<li>Apoptosis occurs in breast cancer cells, human and mouse (Parton)</li>
<li style="text-align:justify"><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">Apoptosis applicable to fishes, hence be used to study as models (dos Santos, N. M., et al. (2008).</span></span></em></li>
<li style="text-align:justify"><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">Apoptosis in humans and baboon ovaries (Kugu, K., et al. (1998)</span></span></em></li>
<li style="text-align:justify"><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">Apoptosis in amphibians during metamorphosis (Ishizuya-Oka, A., et al. (2010).</span></span></em></li>
<li style="text-align:justify"><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">Apoptosis in Drosophila melanogaster (Steller, H. (2008)</span></span></em></li>
<li style="text-align:justify"><em><span style="font-family:Arial,Helvetica,sans-serif">Apoptosis is a highly conserved and essential process across a broad taxonomic range, from unicellular eukaryotes to complex multicellular animals, it is also evident in metazoans (Suraweera, C. D., et al. (2022).</span></em></li>
All stages. Especially critical during embryonic development and in maintaining adult tissue homeostasis.</span></em></p>
</li>
</ul>
<p> </p>
<p> </p>
<h4>Key Event Description</h4>
<p>Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Elmore, 2007]. Apoptosis, also called “physiological cell death”, is involved in cell turnover, physiological involution, and atrophy of various tissues and organs [Kerr et al., 1972]. The formation of apoptotic bodies involves marked condensation of both nucleus and cytoplasm, nuclear fragmentation, and separation of protuberances [Kerr et al., 1972]. Apoptosis is characterized by DNA ladder and chromatin condensation. Several stimuli such as hypoxia, nucleotides deprivation, chemotherapeutical drugs, DNA damage, and mitotic spindle damage induce p53 activation, leading to p21 activation and cell cycle arrest [Pucci et al., 2000]. The SAHA or TSA treatment on neonatal human dermal fibroblasts (NHDFs) for 24 or 72 hrs inhibited proliferation of the NHDF cells [Glaser et al., 2003]. Considering that the acetylation of histone H4 was increased by the treatment of SAHA for 4 hrs, histone deacetylase inhibition may be involved in the inhibition of the cell proliferation [Glaser et al., 2003]. The impaired proliferation was observed in HDAC1<sup>-/-</sup> ES cells, which was rescued with the reintroduction of HDAC1 [Zupkovitz et al., 2010]. An AOP focuses existes on p21 pathway leading to apoptosis, however, alternative pathways such as NF-kappaB signaling pathways may be involved in the apoptosis of spermatocytes [Wang et al., 2017].</p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">Apoptosis is defined as a </span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">programmed cell death</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">. </span></span></span><span style="font-family:"Times New Roman",serif"><span style="color:black"> A decrease in apoptosis or a resistance to cell death is noted is described as a hallmark of cancer by Hanahan et al. It is widely admitted as an essential step in tumor proliferation (Adams, Lowe). </span></span></span></span><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">Apoptosis occurs after activation of a number of intrinsic and extrinsic signals which activate the protease caspase system which in turn activates the destruction of the cell. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">Apoptosis is defined as a </span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">programmed cell death</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">. </span></span></span><span style="font-family:"Times New Roman",serif"><span style="color:black"> A decrease in apoptosis or a resistance to cell death is noted is described as a hallmark of cancer by Hanahan et al. It is widely admitted as an essential step in tumor proliferation (Adams, Lowe). </span></span></span></span><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">Apoptosis occurs after activation of a number of intrinsic and extrinsic signals which activate the protease caspase system which in turn activates the destruction of the cell. </span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:medium"><span style="color:#000000">In mammals, the foetal ovary produces hundreds of thousands of oocytes. But most of them die before birth due to apoptosis (Kaur, S., & Kurokawa, M., 2023). The apoptotic process has a specific pattern at different stages: in foetal ovaries, the majority of apoptotic activity was found in germ cells, whereas in adult quiescent cortical follicles, apoptosis occurred from both granulosa and oocyte cells. The oocyte has been shown to be the one that triggers the apoptotic process and causes follicular atresia (Jin, X., et al. (2011). In humans, the primordial follicles' ovarian endowment is formed throughout foetal development. Apoptotic cell death, which is carried out with the assistance of multiple players and routes conserved from worms to humans, depletes this endowment by at least two-thirds prior to birth. As of right now, apoptosis has been linked to atresia, oocyte loss/selection, folliculogenesis, and oogenesis (Hussein MR, 2005)</span></span></span></em></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">The Bcl-2 is a protein family suppressing apoptosis by <span style="background-color:white">binding and inhibiting</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> two proapoptotic proteins (Bax and Bak)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> and transferring them to the mitochondrial outer membrane. In the absence of inhibition by Bcl2, Bax and Bak destroy the mitochondrial membrane and releases </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">proapoptotic signaling proteins, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">such as</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> cytochrome </span></span></span><em>c</em><em> </em><em><span style="background-color:white"><span style="color:black">which activated the caspase system. </span></span></em><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">An increased</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> expression of </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">these </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">antiapoptotic </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">proteins</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> (Bcl-2, Bcl-x</span></span></span><sub>L</sub>) <em><span style="background-color:white"><span style="color:black">occurs in cancer (Hanahan, Adams, Lowe). Several others pathways such as the l</span></span></em><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">oss of TP53 tumor suppressor function,</span></span></span> or <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">the increase </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">of survival signals (Igf1/2), </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">or decrease of</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">proapoptotic factors (Bax, Bim, Puma)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> can also increase tumor growth <em>(Hanahan, Juntilla).</em></span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">In breast cancer a decrease in apoptosis and a resistance to cell death has been described thoroughly, especially using a dysregulation of the Bcl2 system or TP53 (Parton, </span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Williams</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Shahbandi</span></span></span><span style="font-family:"Times New Roman",serif"><span style="color:black">).</span></span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Apoptosis is characterized by many morphological and biochemical changes <span style="color:black">such as homogenous condensation of chromatin to one side or the periphery of the nuclei, membrane blebbing and formation of apoptotic bodies with fragmented nuclei, DNA fragmentation, enzymatic activation of pro-caspases, or phosphatidylserine translocation that can be measured using electron and cytochemical optical microscopy, proteomic and genomic methods, and spectroscopic techniques [Archana et al., 2013; Martinez et al., 2010; Taatjes et al., 2008; Yasuhara et al., 2003].</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・<span style="color:black">DNA fragmentation can be quantified with comet assay using electrophoresis, where the tail length, head size, tail intensity, and head intensity of the comet are measured [Yasuhara et al., 2003].</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Apoptotic nucleosomes are detected using Cell Death Detection ELISA kit, which was calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Cleavage of PARP is detected with Western blotting [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm [Wu<span style="color:black"> et al.</span>, 2016].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Apoptosis is measured with Annexin V-FITC probes, and the relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry [Wu et al., 2016].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Apoptosis is detected with the Terminal dUTP Nick End-Labeling (TUNEL) method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks [Kressel and Groscurth, 1994].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・For the detection of apoptosis, the testes are fixed in neutral buffered formalin and embedded in paraffin. Germ cell death is visualized in testis sections by Terminal dUTP Nick End-Labeling (TUNEL) staining method [Wade et al., 2008]. The incidence of TUNEL-positive cells is expressed as the number of positive cells per tubule examined for one entire testis section per animal [Wade et al., 2008]</span></span></p>
<h4>References</h4>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Archana, M. et al. (2013), "Various methods available for detection of apoptotic cells", Indian J Cancer 50:274-283</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Elmore, S. (2007), "Apoptosis: a review of programmed cell death", Toxicol Pathol 35:495-516</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Kerr, J.F.R. et al. (1972), "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer 26:239-257</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Kressel, M. and Groscurth, P. (1994), "Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA", Cell Tissue Res 278:549-556</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Martinez, M.M. et al. (2010), "Detection of apoptosis: A review of conventioinal and novel techniques", Anal Methods 2:996-1004</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Pucci, B. et al. (2000), "Cell cycle and apoptosis", Neoplasia 2:291-299</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Taatjes, D.J. et al. (2008), "Morphological and cytochemical determination of cell death by apoptosis", Histochem Cell Biol 129:33-43</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wu, R. et al. (2016), "microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs", PLoS One 11:e0167052</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">Yasuhara, S. et al. (2003), </span>"<span style="color:black">Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis</span>"<span style="color:black">, J Histochem Cytochem 51:873-885</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181</span></span></p>
<p> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. </span></span></span><em>Nature</em> <strong>432</strong>, 307–315 (2004). <a href="https://doi.org/10.1038/nature03098" style="color:#467886; text-decoration:underline"><span style="color:black">https://doi.org/10.1038/nature03098</span></a></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. </span></span></span><em>Nature</em> <strong>432</strong>, 307–315 (2004). <a href="https://doi.org/10.1038/nature03098" style="color:#467886; text-decoration:underline"><span style="color:black">https://doi.org/10.1038/nature03098</span></a></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p>Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.</p>
<p> </p>
<ul>
<li><em><span style="font-family:Tahoma,Geneva,sans-serif">Kaur S, Kurokawa M. Regulation of Oocyte Apoptosis: A View from Gene Knockout Mice. Int J Mol Sci. 2023;24(2).</span></em></li>
<li><em><span style="font-family:Tahoma,Geneva,sans-serif">Jin X, Xiao LJ, Zhang XS, Liu YX. Apotosis in ovary. Front Biosci (Schol Ed). 2011;3(2):680-97.</span></em></li>
<li><em><span style="font-family:Tahoma,Geneva,sans-serif">Hussein MR. Apoptosis in the ovary: molecular mechanisms. Hum Reprod Update. 2005;11(2):162-77.</span></em></li>
<li><em><span style="font-family:Tahoma,Geneva,sans-serif">dos Santos NM, do Vale A, Reis MI, Silva MT. Fish and apoptosis: molecules and pathways. Curr Pharm Des. 2008;14(2):148-69.</span></em></li>
<li><em><span style="font-family:Tahoma,Geneva,sans-serif">Kugu K, Ratts VS, Piquette GN, Tilly KI, Tao XJ, Martimbeau S, et al. Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary. Cell Death Differ. 1998;5(1):67-76.</span></em></li>
<li><em><span style="font-family:Tahoma,Geneva,sans-serif">Ishizuya-Oka A, Hasebe T, Shi YB. Apoptosis in amphibian organs during metamorphosis. Apoptosis. 2010;15(3):350-64.</span></em></li>
<li><em><span style="font-family:Tahoma,Geneva,sans-serif">Steller H. Regulation of apoptosis in Drosophila. Cell Death & Differentiation. 2008;15(7):1132-8.</span></em></li>
<li><em><span style="font-family:Tahoma,Geneva,sans-serif">Suraweera CD, Banjara S, Hinds MG, Kvansakul M. Metazoans and Intrinsic Apoptosis: An Evolutionary Analysis of the Bcl-2 Family. International Journal of Molecular Sciences. 2022;23(7):3691.</span></em></li>
<td><a href="/aops/12">Aop:12 - 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</a></td>
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<td><a href="/aops/48">Aop:48 - Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.</a></td>
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<td><a href="/aops/281">Aop:281 - Acetylcholinesterase Inhibition Leading to Neurodegeneration</a></td>
<td>AdverseOutcome</td>
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<td><a href="/aops/374">Aop:374 - Binding of Sars-CoV-2 spike protein to ACE 2 receptors expressed on brain cells (neuronal and non-neuronal) leads to neuroinflammation resulting in encephalitis</a></td>
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<td><a href="/aops/450">Aop:450 - Inhibition of AChE and activation of CYP2E1 leading to sensory axonal peripheral neuropathy and mortality</a></td>
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<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
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<td><a href="/aops/471">Aop:471 - Various neuronal effects induced by elavl3, sox10, and mbp</a></td>
<td><a href="/aops/471">Aop:471 - Neuron defect induced early behavioral change</a></td>
<p>The necrotic and apoptotic cell death pathways are quite well conserved throughout taxa (Blackstone and Green, 1999, Aravind et al., 2001). It has been widely suggested that apoptosis is also conserved in metazoans, although despite conservation of Bcl-2 proteins, APAF-1, and caspases there is no biochemical evidence of the existence of the mitochondrial pathway in either C. elegans or Drosophila apoptosis (Baum et al., 2007; Blackstone and Green, 1999).</p>
<h4>Key Event Description</h4>
<p style="margin-left:7.0pt">The term neurodegeneration is a combination of two words - "neuro," referring to nerve cells and "degeneration," referring to progressive damage. The term "neurodegeneration" can be applied to several conditions that result in the loss of nerve structure and function, and neuronal loss by necrosis and/or apoptosis</p>
<p>Neurodegeneration is a key aspect of a large number of diseases that come under the umbrella of “neurodegenerative diseases" including Huntington's, Alzheimer’s and Parkinson’s disease. All of these conditions lead to progressive brain damage and neurodegeneration.</p>
<p>Alzheimer's disease is characterised by loss of neurons and synapses in the cerebral cortex and certain subcortical regions, with gross atrophy of the affected regions; symptoms include memory loss.</p>
<p>Parkinson's disease (PD) results from the death of dopaminergic neurons in the midbrain substantia nigra pars compacta; symptoms include bradykinesia, rigidity, and resting tremor.</p>
<p>Several observations suggest correlative links between environmental exposure and neurodegenerative diseases, but only few suggest causative links:</p>
<p>Only an extremely small proportion (less than 5%) of neurodegenerative diseases are caused by genetic mutations (Narayan and Dragounov, 2017). The remainders are thought to be caused by the following:</p>
<p style="margin-left:68.05pt"><!--[if !supportLists]--><span style="font-family:symbol">· </span><!--[endif]-->A build up of toxic proteins in the brain (Evin et al., 2006)</p>
<p style="margin-left:68.05pt"><!--[if !supportLists]--><span style="font-family:symbol">· </span><!--[endif]-->A loss of mitochondrial function that leads to the oxidative stress and creation of neurotoxic molecules that trigger cell death (apoptotic, necrotic or autophagy) (Cobley et al., 2018)</p>
<p style="margin-left:68.05pt"><!--[if !supportLists]--><span style="font-family:symbol">· </span><!--[endif]-->Changes in the levels and activities of neurotrophic factors (Kazim and Iqbal, 2016; Machado et al., 2016; Rodriguez et al., 2014)</p>
<p style="margin-left:68.05pt"><!--[if !supportLists]--><span style="font-family:symbol">· </span><!--[endif]-->Variations in the activity of neural networks (Greicius and Kimmel, 2012)</p>
<p><strong>Protein aggregation</strong>: the correlation between neurodegenerative disease and protein aggregation in the brain has long been recognised, but a causal relationship has not been unequivocally established (Lansbury et al., 2006; Kumar et al., 2016). The dynamic nature of protein aggregation mean that, despite progress in understanding its mechanisms, its relationship to disease is difficult to determine in the laboratory.</p>
<p>Nevertheless, drug candidates that inhibit aggregation are now being tested in the clinic. These have the potential to slow the progression of Alzheimer's disease, Parkinson's disease and related disorders and could, if administered pre-symptomatically, drastically reduce the incidence of these diseases.</p>
<p><strong>Loss of mitochondrial function</strong>: many lines of evidence suggest that mitochondria have a central role in neurodegenerative diseases (Lin and Beal, 2006). Mitochondria are critical regulators of cell death, a key feature of neurodegeneration. Dysfunction of mitochondria induces oxidative stress, production of free radicals, calcium overload, and mutations in mitochondrial DNA that contribute to neurodegenerative diseases. In all major examples of these diseases there is strong evidence that mitochondrial dysfunction occurs early and acts causally in disease pathogenesis. Moreover, an impressive number of disease- specific proteins interact with mitochondria. Thus, therapies targeting basic mitochondrial processes, such as energy metabolism or free-radical generation, or specific interactions of disease-related proteins with mitochondria, hold great promise.</p>
<p style="margin-left:7.0pt"><strong>Decreased level of neurotrophic factors</strong>: decreased levels and activities of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), have been described in a number of neurodegenerative disorders, including Huntington's disease, Alzheimer disease and Parkinson disease (Zuccato and Cattaneo, 2009). These studies have led to the development of experimental strategies aimed at increasing BDNF levels in the brains of animals that have been genetically altered to mimic the aforementioned human diseases, with a view to ultimately influencing the clinical treatment of these conditions. Therefore BDNF treatment is being considered as a beneficial and feasible therapeutic approach in the clinic.</p>
<p style="margin-left:7.0pt"><strong>Variations in the activity of neural networks</strong>: Patients with various neurodegenerative disorders show remarkable fluctuations in neurological functions, even during the same day (Palop et al., 2006). These fluctuations cannot be caused by sudden loss or gain of nerve cells. Instead, it is likely that they reflect variations in the activity of neural networks and, perhaps, chronic intoxication by abnormal proteins that the brain is only temporarily able to overcome.</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="color:#0070c0">Neurodegeneration in relation to COVID19 </span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">SARS-CoV-2 patients present elevated plasma levels of neurofilament light chain protein (NfL), which is a well-known biochemical indicator of neuronal injury (Kanberg et al., 2020). Postmortem brain autopsies demonstrate virus invasion to different brain regions, including the hypothalamus and olfactory bulb, accompanied by neural death and demyelination (Archie and Cucullo 2020; Heneka et al. 2020).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Autopsy results of patients with SARS showed ischemic neuronal damage and demyelination; viral RNA was detected in brain tissue, particularly accumulating in and around the hippocampus (Gu et al. 2005).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Brain magnetic resonance imaging (MRI) investigations in SARS-CoV-2 patients show multifocal hyperintense white matter lesions and cortical signal abnormalities (particularly in the medial temporal lobe) on fluid-attenuated inversion recovery (FLAIR), along with intracerebral hemorrhagic and microhemorrhagic lesions, and leptomeningeal enhancement (Kandemirli et al. 2020; Kremer et al. 2020; Mohammadi et al., 2020).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Moreover, eight COVID-19 patients with signs of encephalopathy had anti–SARS-CoV-2 antibodies in their CSF, and 4 patients had CSF positive for 14-3-3-protein suggesting ongoing neurodegeneration (Alexopoulos et al. 2020).</span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p>The assays for measurements of necrotic or apoptotic cell death are described in the Key Event: Cell injury/Cell death</p>
<p>Recent neuropathological studies have shown that Fluoro-Jade, an anionic fluorescent dye, is a good marker of degenerating neurons. Fluoro-Jade and Fluoro-Jade B were found to stain all degenerating neurons, regardless of specific insult or mechanism of cell death (Schmued et al., 2005). More recently, Fluoro-Jade C was shown to be highly resistant to fading and compatible with virtually all histological processing and staining protocols (Schmued et al., 2005). In addition, Fluoro-Jade C is a good tool for detecting acutely and chronically degenerating neurons (Ehara and Ueda, 2009).</p>
<h4>Regulatory Significance of the AO</h4>
<p>Currently the four available OECD Test Guidelines (TGs) for neurotoxicity testing are entirely based on in vivo neurotoxicity studies: (1)Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure (TG 418); (2) Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study (TG 419); (3) Neurotoxicity Study in Rodents (TG 424) involves daily oral dosing of rats for acute, subchronic, or chronic assessments (28 days, 90 days, or one year or longer); (4) Developmental Neurotoxicity (DNT) Study (TG 426) evaluates in utero and early postnatal effects by daily dosing of at least 60 pregnant rats from implantation through lactation. One of the endpoints required by all four of these OECD TGs is evaluation of neurodegeneration that, so far, is performed through in vivo neuropathological and histological studies. Therefore, neurodegeneration described in this AOP as a key event, has a regulatory relevance and could be performed using in vitro assays that allow a reliable evaluation of neurodegeneration using a large range of existing assays, specific for apoptosis, necrosis and autophagy ( see also KE Cell injury/Cell death).</p>
<h4>References</h4>
<p>Aravind, L., Dixit, V. M., and Koonin, E. V. (2001). Apoptotic Molecular Machinery: Vastly Increased Complexity in Vertebrates Revealed by Genome Comparisons. Science 291, 1279-1284.</p>
<p>Baum, J. S., Arama, E., Steller, H., and McCall, K. (2007). The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis. Cell Death Differ 14, 1508-1517.</p>
<p>Blackstone, N. W., and Green, D. R. (1999). The evolution of a mechanism of cell suicide. Bioessays 21, 84-88.</p>
<p>Cobley JN, Fiorello ML, Bailey DM (2018) 13 reasons why the brain is susceptible to oxidative stress. Redox Biol 15: 490-503</p>
<p>Ehara A, Ueda S. 2009. Application of Fluoro-Jade C in acute and chronic neurodegeneration models: utilities and staining differences. Acta histochemica et cytochemica 42(6): 171-179.</p>
<p>Evin G, Sernee MF, Masters CL (2006) Inhibition of gamma-secretase as a therapeutic intervention for Alzheimer's disease: prospects, limitations and strategies. CNS Drugs 20: 351-72</p>
<p>Kazim SF, Iqbal K (2016) Neurotrophic factor small-molecule mimetics mediated neuroregeneration and synaptic repair: emerging therapeutic modality for Alzheimer's disease. Mol Neurodegener 11: 50</p>
<p>Kumar V, Sami N, Kashav T, Islam A, Ahmad F, Hassan MI (2016) Protein aggregation and neurodegenerative diseases: From theory to therapy. Eur J Med Chem 124: 1105-1120</p>
<p>Lansbury1 PT & Lashuel HA (2006) A century-old debate on protein aggregation and neurodegeneration enters the clinic. Nature 443, 774-779.</p>
<p>Lin1 MT & Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787-795</p>
<p>Machado V, Zoller T, Attaai A, Spittau B (2016) Microglia-Mediated Neuroinflammation and Neurotrophic Factor-Induced Protection in the MPTP Mouse Model of Parkinson's Disease-Lessons from Transgenic Mice. Int J Mol Sci 17</p>
<p>Narayan P, Dragunow M (2017) Alzheimer's Disease and Histone Code Alterations. Adv Exp Med Biol 978: 321-336</p>
<p>Palop JJ, Chin1 J & Mucke L, Review Article A network dysfunction perspective on neurodegenerative diseases. 2006, Nature 443, 768-773</p>
<p>Rodrigues TM, Jeronimo-Santos A, Outeiro TF, Sebastiao AM, Diogenes MJ (2014) Challenges and promises in the development of neurotrophic factor-based therapies for Parkinson's disease. Drugs Aging 31: 239-61</p>
<p>Schmued LC, Stowers CC, Scallet AC, Xu L. 2005. Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons. Brain Res 1035(1): 24-31.</p>
<p>Zuccato C & Cattaneo E, Brain-derived neurotrophic factor in neurodegenerative diseases.2009, Nature Reviews Neurology 5, 311-3</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="color:#0070c0">COVID19-related references relevant to KE Neurodegeneration:</span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Alexopoulos et al. Anti-SARS-CoV-2 antibodies in the CSF, blood-brain barrier dysfunction, and neurological outcome: Studies in 8 stuporous and comatose patients. Neurol Neuroimmunol Neuroinflamm. 2020 Sep 25;7(6):e893.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Archie SR, Cucullo L. Cerebrovascular and neurological dysfunction under the threat of COVID-19: is there a comorbid role for smoking and vaping? Int J Mol Sci. 2020 21(11):3916 12. </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gu J et al. Multiple organ infection and the pathogenesis of SARS. J Exp Med. 2005;202:415–424.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Heneka MT, et al. Immediate and long-term consequences of COVID-19 infections for the development of neurological disease. Alzheimers Res Ther. 2020 12(1):1–3.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Kandemirli SG, et al. Brain MRI findings in patients in the intensive care unit with COVID-19 infection. Radiology. 2020 Oct;297(1):E232-E235.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Kanberg N, et al. Neurochemical evidence of astrocytic and neuronal injury commonly found in COVID-19. Neurology. 2020 Sep 22;95(12):e1754-e1759.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Kremer S, et al. Brain MRI findings in severe COVID-19: a retrospective observational study. Radiology. 2020 Nov;297(2):E242-E251.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Mohammadi S. et al. Understanding the Immunologic Characteristics of Neurologic Manifestations of SARS-CoV-2 and Potential Immunological Mechanisms. Mol Neurobiol. 2020 Dec;57(12):5263-5275.</span></span></span></p>
<h3>List of Adverse Outcomes in this AOP</h3>
<h4><a href="/events/341">Event: 341: Impairment, Learning and memory</a></h4>
<h5>Short Name: Impairment, Learning and memory</h5>
<td><a href="/aops/13">Aop:13 - Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/48">Aop:48 - Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/54">Aop:54 - Inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/77">Aop:77 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony death/failure 1</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/78">Aop:78 - Nicotinic acetylcholine receptor activation contributes to abnormal role change within the worker bee caste leading to colony death failure 1</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/87">Aop:87 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony loss/failure </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/88">Aop:88 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony loss/failure via abnormal role change within caste</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/89">Aop:89 - Nicotinic acetylcholine receptor activation followed by desensitization contributes to abnormal foraging and directly leads to colony loss/failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/90">Aop:90 - Nicotinic acetylcholine receptor activation contributes to abnormal roll change within the worker bee caste leading to colony loss/failure 2</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/12">Aop:12 - 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</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/99">Aop:99 - Histamine (H2) receptor antagonism leading to reduced survival</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/17">Aop:17 - 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</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/442">Aop:442 - Binding to voltage gate sodium channels during development leads to cognitive impairment </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/475">Aop:475 - Binding of chemicals to ionotropic glutamate receptors leads to impairment of learning and memory via loss of drebrin from dendritic spines of neurons</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/483">Aop:483 - Deposition of Energy Leading to Learning and Memory Impairment</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/490">Aop:490 - Co-activation of IP3R and RyR leads to socio-economic burden through reduced IQ and non-cholinergic mechanisms</a></td>
<td><a href="/aops/490">Aop:490 - Co-activation of IP3R and RyR leads to reduced IQ and increased socio-economic burden through non-cholinergic mechanisms</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/499">Aop:499 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via disrupted neurotransmitter release</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/520">Aop:520 - Retinoic acid receptor agonism during neurodevelopment leading to impaired learning and memory</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/525">Aop:525 - Reduced oligodendrocyte differentiation during neurodevelopment leading to impaired learning and memory</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/533">Aop:533 - Retinoic acid receptor antagonism during neurodevelopment leading to impaired learning and memory</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/535">Aop:535 - Binding and activation of GPER leading to learning and memory impairments</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/610">Aop:610 - Decreased thyroid hormone levels in the brain regulated via transport, metabolism and TR activation leading to decreased cognition and motor function</a></td>
<p>Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans. Recently, larval zebrafish has also been suggested as a model for the study of learning and memory (Roberts et al., 2013).</p>
<p>Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans. Recently, larval zebrafish has also been suggested as a model for the study of learning and memory (Roberts et al., 2013). </p>
<p><span style="color:#3498db"><strong>Life stage applicability: </strong>This key event is applicable to various life stages such as during brain development and maturity (Hladik & Tapio, 2016). </span></p>
<p><strong>Life stage applicability</strong>: This key event is applicable to various life stages such as during brain development and maturity (Hladik & Tapio, 2016). </p>
<p><span style="color:#3498db"><strong>Sex applicability:</strong>This key event is not sex specific (Cekanaviciute et al., 2018), although sex-dependent cognitive outcomes have been recently ; Parihar et al., 2020). </span></p>
<p><strong>Sex applicability:</strong>This key event is not sex specific (Cekanaviciute et al., 2018), although sex-dependent cognitive outcomes have been recently ; Parihar et al., 2020). </p>
<p><span style="color:#3498db"><strong>Evidence for perturbation by a prototypic stressor:</strong>Current literature provides ample evidence of impaired learning and memory being induced by ionizing radiation (Cekanaviciute et al., 2018; Hladik & Tapio, 2016). </span></p>
<p><strong>Evidence for perturbation by a prototypic stressor:</strong>Current literature provides ample evidence of impaired learning and memory being induced by ionizing radiation (Cekanaviciute et al., 2018; Hladik & Tapio, 2016). </p>
<p><span style="color:#3498db">Learning can be defined as the process by which new information is acquired to establish knowledge by systematic study or by trial and error (Ono, 2009). Two types of learning are considered in neurobehavioral studies: a) associative learning and b) non- associative learning. Associative learning is based on making associations between different events. In associative learning, a subject learns the relationship among two different stimuli or between the stimulus and the subject’s behavior. On the other hand, non-associative learning can be defined as an alteration in the behavioral response that occurs over time in response to a single type of stimulus. Habituation and sensitization are some examples of non-associative learning. </span></p>
<p>Learning can be defined as the process by which new information is acquired to establish knowledge by systematic study or by trial and error (Ono, 2009). Two types of learning are considered in neurobehavioral studies: a) associative learning and b) non-associative learning. Associative learning is based on making associations between different events. In associative learning, a subject learns the relationship among two different stimuli or between the stimulus and the subject’s behaviour. On the other hand, non-associative learning can be defined as an alteration in the behavioural response that occurs over time in response to a single type of stimulus. Habituation and sensitization are some examples of non-associative learning.</p>
<p><span style="color:#3498db">The memory formation requires acquisition, retention and retrieval of information in the brain, which is characterized by the non- conscious recall of information (Ono, 2009). There are three main categories of memory, including sensory memory, short-term or working memory (up to a few hours) and long-term memory (up to several days or even much longer). </span></p>
<p>The memory formation requires acquisition, retention and retrieval of information in the brain, which is characterised by the non-conscious recall of information (Ono, 2009). There are three main categories of memory, including sensory memory, short-term or working memory (up to a few hours) and long-term memory (up to several days or even much longer).</p>
<p><span style="color:#3498db">Learning and memory depend upon the coordinated action of different brain regions and neurotransmitter systems constituting functionally integrated neural networks (D’Hooge and DeDeyn, 2001). Among the many brain areas engaged in the acquisition of, or retrieval of, a learned event, the hippocampal-based memory systems have received the most study. For example, the hippocampus has been shown to be critical for spatial-temporal memory, visio-spatial memory, verbal and narrative memory, and episodic and autobiographical memory (Burgess et al., 2000; Vorhees and Williams, 2014). However, there is substantial evidence that fundamental learning and memory functions are not mediated by the hippocampus alone but require a network that includes, in addition to the hippocampus, anterior thalamic nuclei, mammillary bodies cortex, cerebellum and basal ganglia (Aggleton and Brown, 1999; Doya, 2000; Mitchell et al., 2002, Toscano and Guilarte, 2005; Gilbert et al., 2006, 2016). Thus, damage to variety of </span></p>
<p> </p>
<p>Learning and memory depend upon the coordinated action of different brain regions and neurotransmitter systems constituting functionally integrated neural networks (D’Hooge and DeDeyn, 2001). Among the many brain areas engaged in the acquisition of, or retrieval of, a learned event, the hippocampal-based memory systems have received the most study. For example, the hippocampus has been shown to be critical for spatial-temporal memory, visio-spatial memory, verbal and narrative memory, and episodic and autobiographical memory (Burgess et al., 2000; Vorhees and Williams, 2014). However, there is substantial evidence that fundamental learning and memory functions are not mediated by the hippocampus alone but require a network that includes, in addition to the hippocampus, anterior thalamic nuclei, mammillary bodies cortex, cerebellum and basal ganglia (Aggleton and Brown, 1999; Doya, 2000; Mitchell et al., 2002, Toscano and Guilarte, 2005; Gilbert et al., 2006, 2016). Thus, damage to variety of brain structures can potentially lead to impairment of learning and memory. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990).While the prefrontal cortex and frontostriatal neuronal circuits have been identified as the primary sites of higher-order cognition in vertebrates, invertebrates utilize paired mushroom bodies, shown to contain ~300,000 neurons in honey bees (Menzel, 2012; Puig et al., 2014).</p>
<p><span style="color:#3498db">brain structures can potentially lead to impairment of learning and memory. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990).While the prefrontal cortex and frontostriatal neural circuits have been identified as the primary sites of higher-order cognition in vertebrates, invertebrates utilize paired mushroom bodies, shown to contain ~300,000 neurons in honey bees (Menzel, 2012; Puig et al., 2014). </span></p>
<p>For the purposes of this KE (AO), impaired learning and memory is defined as an organism’s inability to establish new associative or non-associative relationships, or sensory, short-term or long-term memories which can be measured using different behavioural tests described below.</p>
<p><span style="color:#3498db">For the purposes of this KE (AO), impaired learning and memory is defined as an organism’s inability to establish new associative or non-associative relationships, or sensory, short-term or long-term memories which can be measured using different behavioral tests described below. </span></p>
<h4>How it is Measured or Detected</h4>
<p><strong>In laboratory animals:</strong> in rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, <span style="color:#3498db">Hebb-Williams maze</span>, passive avoidance and Spontaneous alternation and most commonly, the Morris water maze (MWM). Test of novelty such as novel object recognition, and fear based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. A brief description of these tasks follows.</p>
<p>In laboratory animals: in rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, Hebb-Williams maze, passive avoidance and Spontaneous alternation and most commonly, the Morris water maze (MWM). Test of novelty such as novel object recognition, and fear based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. A brief description of these tasks follows. </p>
<p>1) RAM, Barnes, MWM, <span style="color:#3498db">Hebb-Williams maze </span>are examples of spatial tasks, animals are required to learn the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze), or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014). The <span style="color:#3498db">Hebb-Williams maze measures an animal’s problem solving abilities by providing no spatial cues to find the target (Pritchett & Mulder, 2004).</span></p>
<p>RAM, Barnes, MWM,Hebb-Williams mazeare examples of spatial tasks, animals are required to learn the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze), or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014). The Hebb-Williams maze measures an animal’s problem solving abilities by providing no spatial cues to find the target (Pritchett & Mulder, 2004). </p>
<p>2) Novel Object recognition. This is a simpler task that can be used to probe recognition memory. Two objects are presented to animal in an open field on trial 1, and these are explored. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention – I have seen one of these objects before, but not this one (Cohen and Stackman, 2015).</p>
<p>Novel Object recognition. This is a simpler task that can be used to probe recognition memory. Two objects are presented to animal in an open field on trial 1, and these are explored. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention – I have seen one of these objects before, but not this one (Cohen and Stackman, 2015). </p>
<p>3) Contextual Fear conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event. The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009).</p>
<p>Contextual Fear conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event. The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009). </p>
<p>4) Trace fear conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, a light or a tone) and an aversive stimulus (US, a footshock). The unconditioned response (CR) that is elicited upon delivery of the footshock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2001).</p>
<p>Trace fear conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, a light or a tone) and an aversive stimulus (US, a footshock). The unconditioned response (CR) that is elicited upon delivery of the footshock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2001). </p>
<p><span style="color:#3498db">5) Operant Responding. Performance on operant responding reflects the cortex’ ability to organize processes (Rabin et al., 2002). </span></p>
<p>Operant Responding.Performance on operant responding reflects the cortex’ ability to organize processes (Rabin et al., 2002). </p>
<p><strong>In humans:</strong> A variety of standardized learning and memory tests have been developed for human neuropsychological testing, including children (Rohlman et al., 2008). These include episodic autobiographical memory, perceptual motor tests, short and long term memory tests, working memory tasks, word pair recognition memory; object location recognition memory. Some have been incorporated in general tests of intelligence (IQ) such as the Wechsler Adult Intelligence Scale (WAIS) and the Wechsler. Modifications have been made and norms developed for incorporating of tests of learning and memory in children. Examples of some of these tests include:</p>
<p>In humans: A variety of standardized learning and memory tests have been developed for human neuropsychological testing, including children (Rohlman et al., 2008). These include episodic autobiographical memory, perceptual motor tests, short and long term memory tests, working memory tasks, word pair recognition memory; object location recognition memory. Some have been incorporated in general tests of intelligence (IQ) such as the Wechsler Adult Intelligence Scale (WAIS) and the Wechsler. </p>
<p>1) Rey Osterieth Complex Figure test (RCFT) which probes a variety of functions including as visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006).</p>
<p>Modifications have been made and norms developed for incorporating of tests of learning and memory in children. Examples of some of these tests include: </p>
<p>2) Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1994; Talley, 1986).</p>
<p>Rey Osterieth Complex Figure test (RCFT) which probes a variety of functions including as visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006). </p>
<p>3) Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994).</p>
<p>Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1994; Talley, 1986). </p>
<p>4) Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neurospychological test designed to measure memory functions (Lezak, 1994; Talley, 1986).</p>
<p>Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994). </p>
<p>5) Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2011).</p>
<p>Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neurospychological test designed to measure memory functions (Lezak, 1994; Talley, 1986). </p>
<p>6) Staged Autobiographical Memory Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buy lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014).</p>
<p>Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2011). </p>
<p><span style="color:#3498db">7) Attentional set-shifting (ATSET) task. Measures the ability to relearn cues over various schedules of reinforcement (Heisler et al., 2015).</span></p>
<p> </p>
<p>Staged Autobiographical Memory Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buy lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014). </p>
<p>Attentional set-shifting (ATSET) task.Measures the ability to relearn cues over various schedules of reinforcement (Heisler et al., 2015). </p>
<p>8. Comprehensive developmental inventory for infants and toddlers (CDIIT). The CDIIT was designed and standardized in 1996, and it measures the global, cognitive, language, motor, gross motor, fine motor, social, self-help and behavioral developmental status of children from 3 to 71 months old (Wang et al., 1998).</p>
<p>In Honey Bees: For over 50 years an assay for evaluating olfactory conditioning of the proboscis extension reflex (PER) has been used as a reliable method for evaluating appetitive learning and memory in honey bees (Guirfa and Sandoz, 2012; LaLone et al., 2017). These experiments pair a conditioned stimulus (e.g., an odor) with an unconditioned stimulus (e.g., sucrose) provided immediately afterward, which elicits the proboscis extension (Menzel, 2012). After conditioning, the odor alone will lead to the conditioned PER. This methodology has aided in the elucidation of five types of olfactory memory phases in honey bee, which include early short-term memory, late short-term memory, mid-term memory, early long-term memory, and late long-term memory (Guirfa and Sandoz, 2012). These phases are dependent on the type of conditioned stimulus, the intensity of the unconditioned stimulus, the number of conditioning trials, and the time between trials. Where formation of short-term memory occurs minutes after conditioning and decays within minutes, memory consolidation or stabilization of a memory trace after initial acquisition leads to </p>
<p><strong>In Honey Bees:</strong> For over 50 years an assay for evaluating olfactory conditioning of the proboscis extension reflex (PER) has been used as a reliable method for evaluating appetitive learning and memory in honey bees (Guirfa and Sandoz, 2012; LaLone et al., 2017). These experiments pair a conditioned stimulus (e.g., an odor) with an unconditioned stimulus (e.g., sucrose) provided immediately afterward, which elicits the proboscis extension (Menzel, 2012). After conditioning, the odor alone will lead to the conditioned PER. This methodology has aided in the elucidation of five types of olfactory memory phases in honey bee, which include early short-term memory, late short-term memory, mid-term memory, early long-term memory, and late long-term memory (Guirfa and Sandoz, 2012). These phases are dependent on the type of conditioned stimulus, the intensity of the unconditioned stimulus, the number of conditioning trials, and the time between trials. Where formation of short-term memory occurs minutes after conditioning and decays within minutes, memory consolidation or stabilization of a memory trace after initial acquisition leads to mid-term memory, which lasts 1 d and is characterized by activity of the cAMP-dependent PKA (Guirfa and Sandoz, 2012). Multiple conditioning trials increase the duration of the memory after learning and coincide with increased Ca2+-calmodulin-dependent PKC activity (Guirfa and Sandoz, 2012). Early long-term memory, where a conditioned response can be evoked days to weeks after conditioning requires translation of existing mRNA, whereas late long-term memory requires de novo gene transcription and can last for weeks (Guirfa andSandoz, 2012)."</p>
<p>mid-term memory, which lasts 1 d and is characterized by activity of the cAMP-dependent PKA (Guirfa and Sandoz, 2012). Multiple conditioning trials increase the duration of the memory after learning and coincide with increased Ca2+-calmodulin-dependent PKC activity (Guirfa and Sandoz, 2012). Early long-term memory, where a conditioned response can be evoked days to weeks after conditioning requires translation of existing mRNA, whereas late long-term memory requires de novo gene transcription and can last for weeks (Guirfa andSandoz, 2012)." </p>
<h4>Regulatory Significance of the AO</h4>
<p>A prime example of impairments in learning and memory as the adverse outcome for regulatory action is developmental lead exposure and IQ function in children (Bellinger, 2012). Most methods are well established in the published literature and many have been engaged to evaluate the effects of developmental thyroid disruption. The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 or OECD TG 426) <span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">as well as OECD TG 443 (OECD, 2018)</span></span> both require testing of learning and memory (USEPA, 1998; OECD, 2007) advising to use the following tests passive avoidance, delayed-matching-to-position for the adult rat and for the infant rat, olfactory conditioning, Morris water maze, Biel or Cincinnati maze, radial arm maze, T-maze, and acquisition and retention of schedule-controlled behaviour. These DNT Guidelines have been deemed valid to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009).</p>
<p>A prime example of impairments in learning and memory as the adverse outcome for regulatory action is developmental lead exposure and IQ function in children (Bellinger, 2012). Most methods are well established in the published literature and many have been engaged to evaluate the effects of developmental thyroid disruption. The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 or OECD TG 426) as well as OECD TG 443 (OECD, 2018) both require testing of learning and memory (USEPA, 1998; OECD, 2007) advising to use the following tests passive avoidance, delayed-matching-to-position for the adult rat and for the infant rat, olfactory conditioning, Morris water maze, Biel or Cincinnati maze, radial arm maze, T-maze, and acquisition and retention of schedule-controlled behavior. These DNT Guidelines have been deemed valid to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009). </p>
<p>Also, in the frame of the OECD GD 43 (2008) on reproductive toxicity, learning and memory testing may have potential to be applied in the context of developmental neurotoxicity studies. However, many of the learning and memory tasks used in guideline studies may not readily detect subtle impairments in cognitive function associated with modest degrees of developmental thyroid disruption (Gilbert et al., 2012).</p>
<p>Also, in the frame of the OECD GD 43 (2008) on reproductive toxicity, learning and memory testing may have potential to be applied in the context of developmental neurotoxicity studies. However, many of the learning and memory tasks used in guideline studies may not readily detect subtle impairments in cognitive function associated with modest degrees of developmental thyroid disruption (Gilbert et al., 2012). </p>
<p> </p>
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<p> </p>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2942">Relationship: 2942: Activation of MEK, ERK1/2 leads to Increase, intracellular calcium</a></h4>
<p>Astrocytes are networked together by a series of gap junctions permitting to propagate Ca<sup>2+</sup> waves through the linked network (Lobsiger and Cleveland 2007), and Ca2+-mediated intercellular communication is a mechanism by which astrocytes communicate with each other and modulate the activity of adjacent cells (Verderio et al., 2001). Metal mixture (MM) induced alteration in astrocyte morphology may influence [Ca<sup>2+</sup>]i (Barres et al., 1989); in contrast, an increase in [Ca<sup>2+</sup>]<sub>i</sub> may also play a key role in altering astrocyte cytoskeleton, affecting the glia-neuron interaction (Shelton et al., 2000).</p>
<p>Inhibition of GFAP immunoreactivity by MM in developing brain appears to be caused by astrocyte apoptosis. In primary cultures of astrocytes, our data show that MM synergistically induced apoptosis (Rai and others 2010). This was manifested by the activation of MEK/ERK, followed by the activation of JNK pathways, which then enhanced intracellular Ca2+ levels and subsequently ROS generation.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Empirical Evidence</strong>
<p>We treated the astrocytes with a metal-mixture (MM) of arsenic, cadmium, and lead and observed that the MM triggered [Ca<sup>2+</sup>]<sub>i</sub> release (Rai and others 2010). The [Ca2+]<sub>i</sub> release reached its peak after 30 min of MM treatment. Similarly, MM triggered ROS generation, and the ROS generation reached its peak after 1 h of MM treatment. To investigate whether the [Ca<sup>2+</sup>]<sub>i</sub> release was ROS, ERK1/2, or JNK1/2 –dependent, we incubated the MM-treated astrocytes with an antioxidant (a-tocopherol, 200 lg/ml), PD98059 (10lM), or SP600125 (10lM). a-Tocopherol itself was nontoxic. We observed that PD98059 (10lM) or SP600125 (10lM) suppressed [Ca<sup>2+</sup>]<sub>i</sub> release, but a-tocopherol (200 lg/ml) did not. This suggested that [Ca<sup>2+</sup>]<sub>i</sub> release in MM-treated astrocytes was ERK1/2 and JNK1/2 dependent (Rai and others 2010).</p>
<p>Yael and Breitbart (2015) demonstrated for the first time that mouse sperm ERK1/2 is activated upon ZP addition, and that ERK1/2 mediates the elevation of intracellular Ca2+ in the sperm cell prior to the occurrence of the acrosome reaction. The fact that the acrosome reaction, induced by the Ca<sup>2+</sup>-ionophore A23187, was not inhibited by U0126 suggests that ERK1/2 mediates the acrosome reaction by activating Ca2+ transport into the cell. Direct determination of intracellular [Ca<sup>2+</sup>] revealed that Ca2+ influx induced by EGF or ZP was completely blocked by U0126. Thus, it has been established that the increase in ERK1/2 phosphorylation/activation in response to ZP or by activation of the EGF receptor (EGFR) by EGF, is a key event for intracellular Ca<sup>2+</sup> elevation and the subsequent occurrence of the acrosome reaction (Jaldety et al., 2015).</p>
<p>To examine the relationship between Ca<sup>2+</sup> and Erk1/2 signaling, Levin and Borodinsky (2022) inhibited Mek1/2 with PD0325901 and found that this prevents the injury-induced increase in Ca<sup>2+</sup> activity in cells lateral to the axial musculature across the entire 800 µm-wide region measured. This suggests that injury-induced Erk1/2 activation recruits Ca<sup>2+</sup> activity to promote regeneration of the larval tail. Consistent with recruitment of Ca<sup>2+</sup> activity across a wide region of tail, activated Erk1/2 is also present in at least the posterior 800 µm of stump (Levin et al., 2022). However, unlike Ca<sup>2+</sup> activity, Erk1/2 signaling at 20 mpa is activated in a gradient. This could mean that even the lowest level of Erk1/2 signal measured in 800 µm of amputated tail is sufficient to induce the Ca<sup>2+</sup> response, or that a signal is propagated anteriorly from the cells adjacent to the amputation where injury induces high Erk1/2 activation (Levin et al., 2022).</p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Time-scale</strong>
<p>Exposures were conducted for 2 min, 5 min, 10 min, 30 min, 1 h, 2 h, and 24 h. The [Ca2+]<sub>i</sub> release reached its peak after 30 min of MM treatment (Rai and others 2010).</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>The activity of many protein kinases is modulated by Ca2+ and/or Ca2+/calmodulin either directly (PKC, CaM kinase II) or indirectly (PKA via stimulation of adenylyl cyclase and phosphodiesterase by Ca2+/calmodulin) (Kern et al., 1995). Therefore, the effects of Ca2+ and protein kinases on cytoskeletal proteins and neurite initiation are likely to be mediated, at least in part, by changes in protein phosphorylation (Kern et al., 1995).</p>
<h4>References</h4>
<p>Asit Rai and others, Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601, https://doi.org/10.1093/toxsci/kfq266</p>
<p>Barres, B. A., L. L. Chun, and Corey. "Calcium current in cortical astrocytes: induction by cAMP and neurotransmitters and permissive effect of serum factors." Journal of Neuroscience 9.9 (1989): 3169-3175.</p>
<p>Jaldety, Yael, and Haim Breitbart. "ERK1/2 mediates sperm acrosome reaction through elevation of intracellular calcium concentration." Zygote 23.5 (2015): 652-661.</p>
<p>Kern, Marcey, and Gerald Audesirk. "Inorganic lead may inhibit neurite development in cultured rat hippocampal neurons through hyperphosphorylation." Toxicology and applied pharmacology 134.1 (1995): 111-123.</p>
<p>Levin, Jacqueline B., and Laura N. Borodinsky. "Injury-induced Erk1/2 signaling tissue-specifically interacts with Ca2+ activity and is necessary for regeneration of spinal cord and skeletal muscle." Cell calcium 102 (2022): 102540.</p>
<p>Lobsiger, C. S., and Cleveland, D. W. (2007). Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease. Nat. Neuro-sci. 10, 1355–1360.</p>
<p>Shelton, Marilee K., and Ken D. McCarthy. "Hippocampal astrocytes exhibit Ca2+‐elevating muscarinic cholinergic and histaminergic receptors in situ." Journal of neurochemistry 74.2 (2000): 555-563.</p>
<p>Verderio, Claudia, and Michela Matteoli. "ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-γ." The Journal of Immunology 166.10 (2001): 6383-6391.</p>
<p>One of the better characterized apoptotic cascade pathways has mitochondrial dysfunction as its initiator. Mitochondrial dysfunction initiated by the opening of the mitochondrial transition pore leads to mitochondrial depolarization, release of cytochrome C, activation of a variety of caspases and cleavage of downstream death effector proteins, and ultimately results in apoptotic cell death. While a variety of stimuli can trigger opening of the mitochondrial transition pore and cause apoptosis, a sustained intracellular increase in Ca2+ is one of the better-known triggers (Mattson 2000).</p>
</div>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>Intracellular calcium overload may be related to the mitochondrial dysfunction (Yuan et al., 2013). Mitochondria are vital organelles for cellular metabolism and bioenergetics, but they are also key regulators of cell death (Fantin and Leder 2006). Since mitochondria are the major site of ATP production and mitochondrial ΔΨ is the driving force of ATP synthesis, a breakdown in the mitochondrial ΔΨ could lead to a fall in the ATP levels (Chakraborti et al., 1999). The resulting reduction in cellular ATP levels can disrupt ionic homeostasis which can cause an increase in [Ca<sup>2+</sup>]<sub>i</sub> and subsequent cellular apoptosis/necrosis (Grammatopoulos et al., 2004). Notably, in many (if not all) paradigms of apoptosis, ΔΨm represents the point of no return in the cascade of events that ultimately leads to cell death (Kroemer et al., 2007).</p>
<p>The effector phase of apoptosis involves increased mitochondrial Ca<sup>2+</sup> and oxyradical levels, the formation of permeability transition pores (PTP) in the mitochondrial membrane, and release of cytochrome c into the cytosol (Mattson 2000).</p>
<p>The increase of free radicals and Ca<sup>2+</sup> levels associated to Cd exposure may induce mitochondrial disruption (Fern et al., 1996).</p>
<p>Intracellular calcium homeostasis is very important in maintaining the normal function of the cell, in that variations in the concentration of calcium in cells can determine cell survival or death. For example, a high [Ca<sup>2+</sup>]i can cause disruption of mitochondrial Ca<sup>2</sup>+ equilibrium, which results in reactive oxygen species (ROS) formation due to the stimulation of electron flux along the electron transport chain (ETC) (Chacon and Acosta 1991). Under oxidative stress, mitochondrial Ca<sup>2+</sup> accumulation can switch from a physiologically beneficial process to a cell death signal (Ermak and Davies 2002).</p>
<strong>Empirical Evidence</strong>
<div>
<p>Yuan, Yan, et al. 2013 found that BAPTA-AM significantly blocked disruption of Δψm in cells exposed to Cd (5, 10 and 20 µM) for 12 h. Furthermore, cleavage of caspase-9, caspase-3 and PARP were significantly attenuated by BAPTA-AM, which was in agreement with thier observation that BAPTA-AM profoundly prevented Cd-induced apoptosis and cell death of cerebral cortical neurons. However, increased Bax and decreased Bcl-2 levels were not blocked by BAPTA-AM . These data suggest that calcium-mediated mitochondria-caspase b is involved in Cd-induced apoptosis. Moreover, thier results collectively suggested that Cd-induced apoptosis of cerebral cortical neurons occurs through a calcium-mitochondria signaling pathway (Yuan et al., 2013).</p>
<p>Yuan, Yan, et al. 2013 also noted that reduced expression of Bcl-2 increases the expression of Bax, which results in an overload of Ca<sup>2+</sup> in the mitochondria and promotes the opening of permeability transition pores causing mitochondria to swell, with their outer membranes collapsing and exiting into the cytoplasm, which would consequently trigger apoptosis.</p>
</div>
<h4>References</h4>
<div>
<p>Chacon E, Acosta D (1991) Mitochondrial regulation of superoxide by Ca2+: an alternate mechanism for the cardiotoxicity of doxorubicin. Toxicol Appl Pharmacol 107: 117–128.</p>
<p>Chakraborti T, Das S, Mondal M, Roychoudhury S, Chakraborti S (1999) Oxidant, mitochondria and calcium: an overview. Cell Signal 11: 77–85.</p>
<p>Ermak G, Davies KJ (2002) Calcium and oxidative stress: from cell signaling to cell death. Mol Immunol 38: 713–721.</p>
<p>Fantin VR, Leder P (2006) Mitochondriotoxic compounds for cancer therapy. Oncogene 25: 4787–4797.</p>
<p>Grammatopoulos TN, Johnson V, Moore SA, Andres R, Weyhenmeyer JA (2004) Angiotensin type 2 receptor neuroprotection against chemical hypoxia is dependent on the delayed rectifier K+ channel, Na+/Ca2+ exchanger and Na+/K+ ATPase in primary cortical cultures. Neurosci Res 50: 299–306.</p>
<p>Kroemer G, Galluzzi L, Brenner C (2007) Mitochondrial membrane permeabilization in cell death. Physiol Rev 87: 99–163.</p>
<p>Lidsky, Theodore I., and Jay S. Schneider. Lead neurotoxicity in children: basic mechanisms and clinical correlates. Brain 126.1 (2003): 5-19.</p>
<p>Mattson, M. Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1, 120–130 (2000). https://doi.org/10.1038/35040009.</p>
<p>R. Fern, J.A. Black, B.R. Ransom, S.G. Waxman Cd+-induced injury in CNS white matter J. Neurophysiol., 76 (1996), pp. 3264-3273</p>
<p>Yuan, Yan, et al. "Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway." PloS one 8.5 (2013): e64330.</p>
<p>Mitochondria play a role in stress responses and can produce ROS when damaged. Mitochondria are indeed a major source of ROS (Yuan et al., 2013). ROS production is related to the level of ETC (Fleury et al., 2002); it is elevated when electron transport is reduced, which occurs in pathological situations (Wallace 2005).</p>
</div>
</div>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<div>
<div>
<p>A phenotype that is commonly associated with mitochondrial dysfunction, and in fact with many age-related diseases, is the accumulation of damage attributable to the buildup of reactive oxygen species (Leadsham et al., 2013). Indeed, a vicious cycle of decline in which ROS arising from the mitochondrial electron transport chain (ETC) leads to the damage to mitochondrial DNA and a resultant increase in radical production provides the cornerstone of the much scrutinized free radical theory of aging (Harman 1956). However, ROS also serve as important signaling molecules that can promote longevity in C. elegans (Schulz et al., 2007) and also in yeast (Mesquita et al., 2010).</p>
<p>Alterations in mitochondrial physiology could be involved in programmed cell death (PCD). First, reactive oxygen species (ROS) may participate as effector molecules in PCD (Hockenbery et al., 1993; Kane et al., 1993; Sandstrom et al., 1994).</p>
</div>
</div>
<strong>Empirical Evidence</strong>
<div>
<div>
<p>Lopez et al. (2006) showed that in cortical neurons, cadmium exposure induced cellular death, which was, in part, reversed by vitamin C, an antioxidant agent. The apoptosis produced by cadmium was reversed by vitamin C while the necrosis was not affected by this antioxidant molecule. It also appears that in the apoptotic mechanism mediated by cadmium, but not in the necrotic mechanisms, oxidative stress could be implicated. The ability of cadmium to induce oxidative stress in cortical neurons is aided by the induction of ROS by this cation. Cortical neurons treated with cadmium ions at concentrations between 1 and 100 μM, in either the absence or in the presence of serum in the treatment medium, generated ROS. The induction of ROS in these cells type could be mediated by mitochondria alterations because cadmium produces a breakdown of the mitochondrial membrane potential. The decreases in ATP levels and in the mitochondria membrane potential began at 10 and 50 μM cadmium ion, respectively, while the ROS formation was detected at lower doses (100 nM or 1 μM). These results likely indicate that ROS formation occurs or it is detectable before the toxic events on mitochondrial function that lead to the breakdown in mitochondrial potentials.</p>
<p>Zamzami et al. (1995) concluded that at a final level, the shrinkage of ΔΨ<sub>m</sub><sup>low</sup>HE<sup>+</sup> cells is selectively inhibited by substances that suppress mitochondrial ROS generation (rotenone, ruthenium red), as well as by antioxidants such as the vitamin E derivative trolox, alone or incombination with L-ascorbate, or the radical scavenger N-t-butyl-alpha-phenylnitrone. This observation confirms that ROS are PCD effector molecules. In synthesis, these data indicate that ΔΨ<sub>m</sub> reduction and enhanced mitochondrial ROS generation indeed represent two clearly distinct phases of the preapoptotic process. Only after ΔΨ<sub>m </sub>has dropped are ROS generated and do they participate in the perturbation of mitochondrial membranes, as well as in later manifestations of PCD such as cell shrinkage.</p>
<p>Zamazim et al. (1995) went on to state that reduction in ΔΨ<sub>m</sub> and subsequent KOS hyperproduction are observed in several in vitro models of physiological PCD, i.e., models in which nontoxic agents were used to induce PCD in susceptible target cells: TNF-a in U937 cells and anti-IgM in WEHI 231 pre-B cells, as well as CD3 cross-linking in T cell hybridomas. Ceramide, a second messenger involved in the mediation of some PCD types (Obeid et al., 1993; Haimovitz-Friedman et al., 1994), also causes these effects. In all of these systems, alterations in mitochondrial function precede DNA fragmentation and nuclear DNA loss. Thus, it appears that mitochondrial derangement is a constant feature of PCD occurring independently of the PCD-inducing stimulus.</p>
<p>Zhang et al. (2004) reported that Mn<sup>2+</sup> exposure inhibited the complexes I–IV compared to the control. The inhibition of the respiratory activity by Mn<sup>2+</sup> is accompanied by a substantial increase of ROS production rate. They went on to report that NAC, GSH and vitamin C are effective in the prevention of Mn<sup>2+</sup>-induced ROS production and decreases of complexes I–IV activity in isolated mitochondria. Preventive effects of NAC and GSH reveal that cellular GSH are crucial for protection against Mn<sup>2+</sup>-induced toxicity.</p>
</div>
</div>
<h4>References</h4>
<div>
<div>
<p>E. López, C. Arce, M.J. Oset-Gasque, S. Cañadas, M.P. González Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture Free Radic. Biol. Med., 40 (2006), pp. 940-951</p>
<p>Fleury C, Mignotte B, Vayssiere JL (2002) Mitochondrial reactive oxygen species in cell death signaling. Biochimie 84: 131–141.</p>
<p>Haimovitz-Friedman, A., C.-C. Kan, D. Ehleitner, K.S. Persand, M. McLoughlin, Z. Fuks, and K.N. Kolesnick. 1994. Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis. J. Ex F Med. 180:525-535.</p>
<p>Harman, D. (1956). Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298–300.</p>
<p>Hockenbery, D.M., Z.N. Oltvai, X.-M. Yin, C.L. Milliman, and S.J. Korsmeyer. 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 75:241-251.</p>
<p>Kane, D.J., T.A. Sarafian, K. Anton, H. Hahn, E.B. Gralla, J.S. Valentine,T. Ord, and D.E. Bredesen. 1993. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science (Wash. DC). 262:1274-1277</p>
<p>Leadsham, Jane E., et al. "Loss of cytochrome c oxidase promotes RAS-dependent ROS production from the ER resident NADPH oxidase, Yno1p, in yeast." Cell metabolism 18.2 (2013): 279-286.</p>
<p>Mesquita, A., Weinberger, M., Silva, A., Sampaio-Marques, B., Almeida, B., Leao, C., Costa, V., Rodrigues, F., Burhans, W.C., and Ludovico, P. (2010). Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity. Proc. Natl. Acad. Sci. USA 107, 15123–15128.</p>
<p>Obeid, L.M., C.M. Linardic,L.A. Karolak, and Y.A. Hannun. 1993. Programmed cell death induced by ceramide. Science (Wash. DC). 259:1769-1771.</p>
<p>Sandstrom, P.A., M.D. Mannie, and T.M. Buttke. 1994. Inhibition of activation-induced death in a T cell hybridoma by thiol antioxidants: oxidative stress as a mediator of apoptosis. J. Leukocyte Biol. 55:221-226.</p>
<p>Schulz, T.J., Zarse, K., Voigt, A., Urban, N., Birringer, M., and Ristow, M. (2007). Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab. 6, 280–293.</p>
<p>Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39: 359–407.</p>
<p>Yuan, Yan, et al. "Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway." PloS one 8.5 (2013): e64330.</p>
<p>Zamzami, Naoufal, et al. "Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death." The Journal of experimental medicine 182.2 (1995): 367-377.</p>
<p>Zhang, Surong, Juanling Fu, and Zongcan Zhou. "In vitro effect of manganese chloride exposure on reactive oxygen species generation and respiratory chain complexes activities of mitochondria isolated from rat brain." Toxicology in vitro 18.1 (2004): 71-77.</p>
</div>
</div>
</div>
<div>
<h4><a href="/relationships/2966">Relationship: 2966: Increased, Reactive oxygen species leads to Apoptosis</a></h4>
<h4><a href="/relationships/2966">Relationship: 2966: Increase, ROS leads to Apoptosis</a></h4>
<p>ROS generation in normal cells, including neurons, occurs within homeostatic control. When ROS levels exceed the antioxidant capacity of a cell, a deleterious condition known as oxidative stress occurs (Klein and Ackerman 2003). Unchecked, excessive ROS can lead to the destruction of cellular components including lipids, protein, and DNA, and ultimately cell death via apoptosis or necrosis (Kannan and Jain 2000).</p>
</div>
</div>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<div>
<div>
<p>Reactive oxygen species (ROS) can be derived from exogenous sources or produced in vivo; these include the superoxide anion (O 2), the hydroxyl radical (OH), and hydrogen peroxide (H 2O2). ROS at low levels participate in cell signaling while higher ROS concentrations are deleterious due to the oxidation of proteins, lipids, and DNA. Additionally, persistent ROS production compromises the cellular antioxidant defense systems and results in oxidative stress and apoptosis (337). ROS can initiate apoptosis via the mitochondrial and death receptor pathways. In the former, ROS have been shown to induce loss of the m, release of mitochondrial pro-apoptotic proteins, and activation of caspase 3 (49).</p>
<p>ROS signaling has been shown to mediate cytokine-induced apoptosis (Okouchi et al., 2007). TNF is a pro-inflammatory cytokine produced by macrophages and is the most studied cytokine in apoptosis and the pathophysiology of various diseases, including neurodegenerative disorders (Jackson et al., 1999). Mechanistically, the binding of TNF to its receptor activates the NF-B and JNK signaling pathways believed to be mediated by ROS (Okouchi et al., 2007). A role for ROS has also been implicated in death receptor-mediated apoptosis induced by apoptosis signal-regulating kinase 1 (ASK1), an ubquitiously expressed MAP kinase kinase kinase (MAPKKK), that activates JNK and p38 MAP kinase pathways (Okouchi et al., 2007).</p>
</div>
</div>
<strong>Empirical Evidence</strong>
<div>
<div>
<p>Free radical scavenger or antioxidant N-acetyl-L-cysteine, a thiol-containing compound, has been shown to directly reduce the levels of ROS (Aruoma et al., 1989; Kim and Sharma 2004; Poliandri et al., 2003). To confirm that Cd-induced neuronal apoptosis is indeed due to its induction of ROS generation, PC12 and SH-SY5Y cells were pretreated with NAC (5mM) for 1h, and then exposed to Cd (10 and 20μM) for 24h (Long et al., 2008). Chen et al. (2008) found that NAC dramatically blocked Cd-induced ROS generation in PC12 cells and SH-SY5Y cells. In addition, to further quantify the protective effect of NAC on Cd-induced apoptosis via blockage of ROS in a larger cell population, they performed annexin-V-FITC and propidium iodide staining followed by flow cytometry. They found NAC alone did not affect cell viability. However, it significantly blocked Cd-induced apoptosis.</p>
<p>Asit Rai et al. 2010 found that a metal mixture of arsenic, cadmium, and lead triggered ROS generation, reaching its peak after 1 hour of treatment. They next investigated whether ERK1/2, JNK1/2, [Ca<sup>2+</sup>]<sub>i</sub> and ROS signaling resulted in apoptosis by reating the MM-treated astrocytes with α-tocopherol (200 μg/ml), PD98059 (10μM), BAPTA-AM (5μM), or SP600125 (10μM). They all suppressed apoptosis suggesting that activation of ERK1/2 and JNK1/2, followed by increased [Ca<sup>2+</sup>]<sub>i</sub> and ROS generation, resulted in apoptosis in the MM-treated astrocytes.</p>
<p>When astrocytes were exposed to H2O2 for 30 min and then incubated without H2O2 for 1–5 days, cell toxicity including apoptosis was observed (Kazuhiro et al., 2004). Furthermore, the reperfusion injury induced by Ca<sup>2+</sup> depletion or H2O2 exposure was exacerbated by the catalase inhibitor, 3-amino-1,2,4-triazole, and the GSH synthesis inhibitors, l-buthionine-S,R-sulfoximine and xanthine, while the injury was blocked by GSH, catalase and the iron chelators, 1,10-phenanthroline and deferoxamine (Takuma et al., 1999). These findings indicate that Ca<sup>2+</sup> reperfusion-induced apoptosis is mediated by ROS production, especially by hydroxyl radical formation (Kazuhiro et al., 2004).</p>
<p>Exposure of cells to 5 M iAs significantly triggered the expression of ER stress-related molecules, including: the proteins and mRNAs expression of GRP 78, CHOP, XBP-1 in a time-dependent manner (for 6–24 h) as well as the degradation of full-length (55 kDa) caspase-12 (downstream ER stress molecule). However, GRP 94 was not affected by iAs treatment. These effects of iAs-induced ER stress protein responses could be reversed by pre-treatment with NAC. Furthermore, transfection of Neuro-2a cells with GRP 78- and CHOP-specific si-RNA, respectively, markedly reduced the protein expression levels of GRP 78 and CHOP in the cells treated with iAs and significantly attenuate the iAs-induced caspase-3, -7, and -12 activations. These results indicate that oxidative stress-mediated ER stress activation pathway is also involved in iAs-induced neuronal cell apoptosis (Tien-Hui, et al. 2014).</p>
<p>Recent studies have shown that ROS generation induced by toxic metals (including arsenic) causes neuronal apoptosis, which is closely associated with the progression of neurodegenerative diseases (Bharathi and Jagannathan 2006; Flora et al., 2009; Gharibzadeh 2008).</p>
<p>Okouchi et. al. (2007) found that peroxide-induced apoptosis in undifferentiated PC12 cells was mediated by an early loss of the cellular glutathione–glutathione disulfide (GSH/GSSG) redox balance that preceded an increase in Bax expression, mitochondrial-to-cytosol cytochrome c translocation, and activation of caspase 3 (Pias and Aw 2002; Pias and Aw 2002; Pias et al., 2003). Apoptosis was ameliorated by the overexpression of mitochondrial superoxide dismutase, MnSOD (SOD2), and by pretreatment of cells with the antioxidant, N-acetyl cysteine (NAC) <sup>(23-25)</sup>.</p>
<p>As first demonstrated in mouse fibrosarcoma cells, TNF treatment disrupts mitochondrial electron transport and enhances ROS production <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Schulze–Osthoff et al., 1992)</span></span>. Recent studies by Han et al. (2006) showed that modulation of the hepatocyte redox environment by ROS interfered with NF-B signaling in TNF-induced apoptosis. Notably, cell apoptosis occurred within a certain redox window in which mild redox imbalance inhibited NF-B activation, but not caspase activity <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Okouchi et al., 2007)</span></span>.</p>
</div>
</div>
<strong>Uncertainties and Inconsistencies</strong>
<div>
<div>
<p>ROS and/or oxidative damage can activate gene transcription and transcribed genes may be implicated in either cell survival or cell death (Klein and Ackerman 2003).</p>
<p>The increase in reactive oxygen species at As(III) concentrations of 0.5 mg/l or more may play an apoptogenic role and/or be a consequence of events occurring during apoptosis<span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span>(Rocha et al. 2011). It is generally reported that ROS cause an increase in [Ca<sup>2+</sup>]i of various cell types, which might be one of the causes for the C17.2 cells to enter apoptosis (Rocha et al. 2011). According to Hool and Corry (2007), the redox control of Ca<sup>2+</sup> transport is due to the fact that ROS can react with the thiol groups of protein that form part of the Ca<sup>2+</sup> transporters or channels. Alternatively, mitochondrial matrix Ca<sup>2+</sup> overload can lead to enhanced generation of reactive oxygen species, triggering the permeability transition pore, dissipation of transmembrane mithocondrial potential, and cytochrome c release <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Brookes et al., 2004)</span></span>. In any case, the fact that treatment with various antioxidants (vitamin E, tocopherol, and quercetin) did not rescue the cells from death by apoptosis indicates that oxidative stress was not the main cause of the observed cell death (Rocha et al. 2011).</p>
<p>Superoxides and lipid peroxidation are increased during apoptosis induced by myriad stimuli (Bredesen 1995). However, generation of ROS may be a relatively late event, occurring after cells have embarked on a process of caspase activation (Green and Reed 1998). In this regard, attempts to study apoptosis under conditions of anoxia have demonstrated that at least some proapoptotic stimuli function in the absence or near absence of oxygen, which implies that ROSs are not the sine qua non of apoptosis (Jacobson and Raff 1995). However, ROSs can be generated under conditions of virtual anaerobiosis (Degli Esposti and McLennan 1998), and thus their role in apoptosis cannot be excluded solely on this basis (Green and Reed 1998).</p>
<p>Okouchi et. al. (2007) found that PC12 apoptosis can be initiated by GSH/GSSG redox imbalance alone independently of ROS generation (Pias et al., 2003), suggesting that a loss of cellular redox homeostasis is downstream of ROS signaling in neuronal cell apoptosis.</p>
</div>
</div>
<h4>References</h4>
<div>
<div>
<p>A.H. Poliandri, J.P. Cabilla, M.O. Velardez, C.C. Bodo, B.H. Duvilanski Cadmium induces apoptosis in anterior pituitary cells that can be reversed by treatment with antioxidants Toxicol. Appl. Pharmacol., 190 (2003), pp. 17-24</p>
<p>Asit Rai et al., Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601</p>
<p>Bharathi, Ravid R, Jagannathan Rao KS (2006) Role of metals in neuronal apoptosis: challenges associated with neurodegeneration. Curr Alzheimer Res 3:311–326</p>
<p>Bredesen, Dale E. "Neural apoptosis." Annals of neurology 38.6 (1995): 839-851.</p>
<p>Chen, Long, Lei Liu, and Shile Huang. "Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5." Free Radical Biology and Medicine 45.7 (2008): 1035-1044.</p>
<p>Degli Esposti, Mauro, and Holly McLennan. "Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis." FEBS letters 430.3 (1998): 338-342.</p>
<p>Flora SJ, Bhatt K, Mehta A (2009) Arsenic moiety in gallium arsenide is responsible for neuronal apoptosis and behavioral alterations in rats. Toxicol Appl Pharmacol 240:236–244</p>
<p>Gharibzadeh S, Hoseini SS (2008) Arsenic exposure may be a risk factor for Alzheimer’s disease. J Neuropsychiatr Clin Neurosci 20:501</p>
<p>Green, Douglas R., and John C. Reed. "Mitochondria and apoptosis." science 281.5381 (1998): 1309-1312.</p>
<p>Han D, Hanawa N, Saberi B, and Kaplowitz N. Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis. Free Rad Biol Med 41: 627–639, 2006.</p>
<p>J. Kim, R.P. Sharma Calcium-mediated activation of c-Jun NH2-terminal kinase (JNK) and apoptosis in response to cadmium in murine macrophages Toxicol. Sci., 81 (2004), pp. 518-527</p>
<p>Jackson CE, Fisher RE, Hsu AP, Anderson SM, Choi YN, Wang J, Dale JK, Fleisher TA, Middelton LA, Sneller MC, Leonardo MJ, Straus SE, and Puck JM. Autoimmune lymphoproliferative syndrome with defective Fas: genotype influences penetrance. Am J Hum Genet 64: 1002–1014, 1999</p>
<p>Jacobson, Michael D., and Martin C. Raff. "Programmed cell death and Bcl-2 protection in very low oxygen." Nature 374.6525 (1995): 814-816.</p>
<p>K. Takuma, E. Lee, M. Kidawara, K. Mori, Y. Kimura, A. Baba, T. Matsuda Apoptosis in Ca2+ reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-κB activation Eur. J. Neurosci., 11 (1999), pp. 4204-4212</p>
<p>Klein, Jeffrey A., and Susan L. Ackerman. "Oxidative stress, cell cycle, and neurodegeneration." The Journal of clinical investigation 111.6 (2003): 785-793.</p>
<p>L.C. Hool, B. Corry Redox control of calcium channels: from mechanisms to therapeutic opportunities Antioxid. Redox Signal, 9 (2007), pp. 409-435</p>
<p>Lu, Tien-Hui, et al. "Arsenic induces reactive oxygen species-caused neuronal cell apoptosis through JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-regulated pathways." Toxicology letters 224.1 (2014): 130-140.</p>
<p>O.I. Aruoma, B. Halliwell, B.M. Hoey, J. Butler The antioxidant action of N-acetylcysteine: itS reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid Free Radic. Biol. Med., 6 (1989), pp. 593-597</p>
<p>Okouchi, Masahiro, et al. "Neuronal apoptosis in neurodegeneration." Antioxidants & redox signaling 9.8 (2007): 1059-1096.</p>
<p>P.S. Brookes, Y. Yoon, J.L. Robotham, M.W. Anders, S.-S. Shen Calcium ATP and ROS: a mitochondrial love-hate triangle Am. J. Physiol. Cell Physiol., 287 (2004), pp. 817-833</p>
<p>Pias EK and Aw TY. Apoptosis in mitotic competent undifferentiated cells is induced by cellular redox imbalance independent of reactive oxygen species production. FASEB J 16:781–790, 2002</p>
<p>Pias EK and Aw TY. Early redox imbalance mediates hydroperoxide-induced apoptosis in mitotic competent undifferentiated PC-12 cells. Cell Death Differ 9: 1007–1016, 2002.</p>
<p>Pias EK, Ekshyyan OY, Rhoads CA, Fuseler J, Harrison L, and Aw TY. Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells. J Biol Chem 278: 13294–13301, 2003.</p>
<p>Pias EK, Ekshyyan OY, Rhoads CA, Fuseler J, Harrison L, and Aw TY. Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells. J Biol Chem 278: 13294–13301, 2003</p>
<p>Rocha, R. A., et al. "Arsenic and fluoride induce neural progenitor cell apoptosis." Toxicology letters 203.3 (2011): 237-244.</p>
<p>Schulze–Osthoff K, Bakker AC, Vanhaesebroeck B, Beyaert R, Jacob WA, and Fiers W. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial function. J Biol Chem 267: 5317–5323, 1992.</p>
<p>Takuma, Kazuhiro, Akemichi Baba, and Toshio Matsuda. "Astrocyte apoptosis: implications for neuroprotection." Progress in neurobiology 72.2 (2004): 111-127.</p>
</div>
</div>
</div>
<div>
<h4><a href="/relationships/2967">Relationship: 2967: Apoptosis leads to N/A, Neurodegeneration</a></h4>
<p>In the central nervous system (CNS), neuronal apoptosis is a physiological process that is an integral part of neurogenesis, and aberrant apoptosis has been implicated in the pathogenesis of neurodegeneration (Okouchi et al., 2007).</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>During the development of the nervous system, an excessive number of neurons is produced. This massive overproduction of neurons is followed by a programmed demise of roughly one half of the originally produced cells (Okouchi et al., 2007). The precisely controlled process is referred to as naturally occurring neuronal death which is a highly conserved cellular mechanism in diverse organisms, ranging from invertebrate species such as the nematode (Okouchi et al., 2007), Caenorhabditis elegans, and insects, to nearly all of the studied vertebrate species (Mishima et al., 1999). Natural neuronal death is be lieved to mold the nervous system’s cellular structure and function (Okouchi et al., 2007).</p>
<p>As axons extend, they also bifurcate with each branch forming of its own growth cone, a process that is also regulated by apoptosis (Chen et al., 2020). Under normal conditions, a low level of caspase maintains a balance between growth cone attraction and repulsion and inhibits axon extension; however, in PTSD, apoptosis is enhanced in key brain regions and caspase activation alters growth cone trajectory and dendritic pruning, leading to axon misguidance and dendrite degeneration. The combined outcome of these processes is the formation of fewer or incorrect synapses in PTSD that are defective in information transmission and cause abnormalities in memory and behavior (Chen et al., 2020).</p>
<strong>Empirical Evidence</strong>
<p>Rai, Nagendra Kumar, et al. (2013) concluded that the metal mixture arsenic, lead, and cadmium (a) induced dose-dependent modulation in the expression levels of myelin and axon proteins leading to hypo-myelination in cortex; (b) reduced axon area and myelin density in O.N.; and (c) attenuated RGC-differentiation in retina. Apoptosis in the oligodendrocytes, axonal neurons and RGCs promoted the MM-mediated white matter damage. </p>
<p>Increased apoptosis in MBP and NF damage the white matter of CNS (Petzold et al., 2011; Pun et al., 2011). In consistence, Rai, Nagendra Kumar, et al. (2013) found that the impairment in postnatal oligodendrocytes and axons further increased cellular apoptosis in the brain and O.N. The neuroaxonal degeneration in the retina also involved a rise in apoptosis in Brn3b and NF. During development, Brn3b is crucial for RGC survival, and its apoptosis may affect the expression of several genes linked with axonal integrity and function (Pan, et al., 2005). Therefore, the increased MM-related apoptosis to axonal neurons in the retina could be the fallout of RGC damage (Rai et al., 2013). Altogether, the MM, in all probability, elevates the apoptosis-mediated pruning of the myelinating cells during CNS development (Rai et al., 2013).</p>
<p>In the context of an Alzheimer's disease brain; compelling evidence of apoptotic involvement comes from studies of Rohn et al., (2002) who demonstrated the activation of mitochondrial and receptor-mediated apoptotic pathways in AD hippocampal brain sections wherein active caspase 9 was co-localized with active caspase 8 (Okouchi et al., 2007). Moreover, the distribution of caspase-cleaved fragments of tau suggests that the activation of caspases preceded the formation of neurofibrillary tangles in brains of AD patients (Chiueh et al., 2000). In addition, the intracellular amyloid beta peptide 1-42 (A beta (1-42)) has been shown to induce human neuronal cell apoptosis through Bax activation that resulted in cytochrome c release and activation of caspase 6 (Zhang et al., 2002).</p>
<p>The participation of apoptosis in disease pathogenesis in humans is supported by the demonstration of caspases 1, 3, 8, and 9, and cytochrome c activation in the brains of Huntington Disease patients (Kiechle et al., 2022; Teng et al., 2006; Sanchez et al., 1999).</p>
<p>The involvement of hippocampal neuronal apoptosis in diabetic encephalopathy has been demonstrated in diabetic animal models (Li et al., 2005), and evidence of classical apoptosis was associated with decreased neuronal densities, and learning and cognitive deficits (Sima and Li 2005).</p>
<p>Cognitive impairment in BB/Wor rats is associated with evidence of classical apoptosis in the hippocampus, including DNA fragmentation, positive TUNEL staining, elevated Bax/Bcl-x ratio, increased caspase 3 activities and decreased neuronal densities (Li et al., 2002), common features in diabetic encephalopathy.</p>
<p>Notable among endogenous antioxidants, is estradiol, with proven effectiveness against beta-amyloid-induced neuronal apoptosis in in vitro models of AD and PD (Gandy 2003; Yao et al., 2007). Accelerated beta-amyloid plaque formation in animal models of AD is associated with brain estradiol deficiency (Gandy 2003). Estradiol mediates its effect by binding to the estrogen receptor, and targets a plethora of prosurvival cellular processes (Okouchi et al., 2007). These include neuronal expression of Bcl-2 members, upregulation of antioxidant proteins such as TRX, MnSOD, and nNOS, Akt signaling, and inhibition of transcriptional and apoptotic activity of the APPct complex (Yao et al., 2007; Bao et al., 2007; Chiueh et al., 2003; Koh et al., 2006). Melatonin is another naturally occurring neuroprotectant that decreases amyloid fibril formation (Pappolla et al., 1998) and attenuates neuronal apoptosis in in vitro and animal models of AD and PD (Chiueh et al., 2000; Deigner et al., 2000; Matsubara et al., 2003). Its neuroprotective effects appear to be the result of antioxidant and anti-amyloidogenic properties (Pappolla et al., 2002) and are independent of binding to membrane receptors (Okouchi et al., 2007).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>While the molecular mechanisms underlying neuronal apoptosis and diabetic encephalopathy remain unresolved, it appears that diabetes-associated perturbations in the insulin/IGF system and hyperglycemia may play prominent roles (Li and Sima 2004).</p>
<h4>References</h4>
<p>A. Petzold, et al. In vivo monitoring of neuronal loss in traumatic brain injury: a microdialysis study Brain, 134 (Pt 2) (2011), pp. 464-483</p>
<p>Bao J, Cao C, Zhang X, Jiang F, Nicosia SV, and Bai W. Suppression of beta-amyloid precursor protein signaling into the nucleus by estrogens mediated through complex formation between the estrogen receptor and Fe65. Mol Cell Biol 27: 1321–1333, 2007.</p>
<p>Chen, Xinzhao, et al. "Synapse impairment associated with enhanced apoptosis in post‐traumatic stress disorder." Synapse 74.2 (2020): e22134.</p>
<p>Chiueh C, Lee S, Andoh T, and Murphy D. Induction of antioxidative and antiapoptotic thioredoxin supports neuroprotective hypothesis of estrogen. Endocrine 21: 27–31, 2003.</p>
<p>Chiueh CC, Andoh T, Lai AR, Lai E, and Krishna G. Neuroprotective strategies in Parkinson’s disease: protection against progressive nigral damage induced by free radicals. Neurotox Res 2: 293–310, 2000.</p>
<p>Deigner HP, Haberkorn U, and Kinscherf R. Apoptosis modulators in the therapy of neurodegenerative diseases. Expert Opin Investig Drugs 9: 747–764, 2000.</p>
<p>Gandy S. Estrogen and neurodegeneration. Neurochem Res 28: 1003–1008, 2003.</p>
<p>Kiechle T, Dedeoglu A, Kubilus J, Kowall NW, Beal MF, Friedlander RM, Hersch SM, and Ferrante RJ. Cytochrome C and caspase-9 expression in Huntington’s disease. Neuromolec Med 1: 183–195, 2002.</p>
<p>Koh PO, Won CK, and Cho JH. Estradiol prevents the injury-induced decrease of Akt/glycogen synthase kinase 3beta phosphorylation. Neurosci Lett 404: 303–308, 2006.</p>
<p>L. Pan, et al. Functional equivalence of Brn3 POU-domain transcription factors in mouse retinal neurogenesis Development, 132 (4) (2005), pp. 703-712</p>
<p>Li ZG and Sima AA. C-peptide and central nervous system complications in diabetes. Exp Diabesity Res 5: 79–90, 2004.</p>
<p>Li ZG, Zhang W, and Sima AA. The role of impaired insulin/IGF action in primary diabetic encephalopathy. Brain Res 1037: 12–24, 2005.</p>
<p>Li ZG, Zhang W, Grunberger G, and Sima AA. Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res 946: 221–231, 2002.</p>
<p>Matsubara E, Bryant-Thomas T, Pacheco Quinto J, Henry TL, Poeggeler B, Herbert D, Cruz–Sanchez F, Chyan YJ, Smith MA, Perry G, Shoji M, Abe K, Leone A, Grundke–Ikbal I, Wilson GL, Ghiso J, Williams C, Refolo LM, Pappolla MA, Chain DG, and Neria E. Melatonin increases survival and inhibits oxidative and amyloid pathology in a transgenic model of Alzheimer’s disease. J Neurochem 85: 1101–1108, 2003.</p>
<p>Mishima K, Tozawa T, Satoh K, Matsumoto Y, Hishikawa Y, and Okawa M. Melatonin secretion rhythm disorders in patients with senile dementia of Alzheimer’s type with disturbed sleep-waking. Biol Psychiatry 45: 417–421, 1999</p>
<p>Okouchi, Masahiro, et al. "Neuronal apoptosis in neurodegeneration." Antioxidants & redox signaling 9.8 (2007): 1059-1096.</p>
<p>P.B. Pun, et al. Low level primary blast injury in rodent brain Front. Neurol., 2 (2011), p. 19</p>
<p>Pappolla M, Bozner P, Soto C, Shao H, Robakis NK, Zagorski M, Frangione B, and Ghiso J. Inhibition of Alzheimer beta-fibrillogenesis by melatonin. J Biol Chem 273: 7185–7188, 1998</p>
<p>Pappolla MA, Simovich MJ, Bryant–Thomas T, Chyan YJ, Poeggeler B, Dubocovich M, Bick R, Perry G, Cruz–Sanchez F, and Smith MA. The neuroprotective activities of melatonin against the Alzheimer beta-protein are not mediated by melatonin membrane receptors. J Pineal Res 32: 135–142, 2002.</p>
<p>Rai, Nagendra Kumar, et al. "Exposure to As, Cd and Pb-mixture impairs myelin and axon development in rat brain, optic nerve and retina." Toxicology and applied pharmacology 273.2 (2013): 242-258.</p>
<p>Rohn TT, Rissman RA, Davis MC, Kim YE, Cotman CW, and Head E. Caspase-9 activation and caspase cleavage of tau in the Alzheimer’s disease brain. Neurobiol Dis 11: 341–354, 2002</p>
<p>Sanchez I, Xu CJ, Juo P, Kakizaka A, Blenis J, and Yuan J. Caspase-8 is required for cell death induced by expanded polyglutamine repeats. Neuron 22: 623–633, 1999.</p>
<p>Sima AA and Li ZG. The effect of C-peptide on cognitive dysfunction and hippocampal apoptosis in type 1 diabetic rats. Diabetes 54: 1497–1505, 2005.</p>
<p>Teng X, Sakai T, Liu L, Sakai R, Kaji R, and Fukui K. Attenuation of MPTP-induced neurotoxicity and locomotor dysfunction in Nucling-deficient mice via suppression of the apoptosome pathway. J Neurochem 97: 1126–1135, 2006.</p>
<p>Yao M, Nguyen TV, and Pike CJ. Estrogen regulates Bcl-w and Bim expression: role in protection against beta-amyloid peptide induced neuronal death. J Neurosci 27:1422–1433, 2007.</p>
<p>Zhang Y, McLaughlin R, Goodyer C, and LeBlanc A. Selective cytotoxicity of intracellular amyloid peptide1-42 through p53 and Bax in cultured primary human neurons. J Cell Biol 156: 519–529, 2002.</p>
</div>
<div>
<h4><a href="/relationships/1069">Relationship: 1069: N/A, Neurodegeneration leads to Impairment, Learning and memory</a></h4>
<td><a href="/aops/12">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</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/500">Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
<td>adjacent</td>
<td>Not Specified</td>
<td>Not Specified</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p>Animal models of neurodegenerative diseases, in particular Alzheimer's disease, contributed to the elucidation of the link between amyloid protein and tau hyperphosphorylation and cognitive deficits. Bilateral injections of amyloid-b peptide in the frontal cortex of rats leads to progressive decline in memory and neurodegeneration in hippocampus (for review see Eslamizade et al., 2016). Recent findings have shown that soluble forms of Ab rather than insoluble forms (fibrils and plaques) are associated with memory impairment in early stages of Alzheimer's disease (for review see Salgado-Puga and Pena-Ortega, 2015). Several lines of evidence suggest that the small oligomeric forms of Ab and tau may act synergistically to promote synaptic dysfunction in Alzheimer's disease (for review see Guerrerro-Minoz et al., 2015). Some reports proposed the concept of imbalance between production and clearance of Ab42 and related Ab peptides, as an initiating factor inducing hyperphosphorylation of tau and leading to neuritic dystrophy and synaptic dysfunction (for review see Selkoe and Hardy, 2016). Recent trials of three different antibodies against amyloid peptides have suggested a slowing of cognitive decline in post hoc analyses of mild Alzheimer subjects (for review see Selkoe and Hardy, 2016). Therefore cognitive deficits may be related to the level and extent of classical Alzheimer pathology landmarks, but it is also influenced by neurodegeneration (for review see Braskie and Thompson, 2013). Indeed decreased hippocampal volume due to widespread neurodegeneration and visualized by neuroimaging appears to be a significant predictor of memory decline (for review see Braskie and Thompson, 2016).</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>It is well accepted that impairment of cell function or cell loss in hippocampus will interfere with memory processes, since the hippocampus plays a key role in memory (Barker and Warburton, 2011). In Alzheimer's disease, hippocampus and entorhinal cortex are affected early in the disease process and cognitive deficit is correlated with brain atrophy (for review Braskie and Thompson, 2013).</p>
<strong>Empirical Evidence</strong>
<p><em>Include consideration of temporal concordance here </em></p>
<p>Pre-natal and post-natal Pb exposure affects the hippocampus and the frontal cortex (Schneider et al., 2012). Rats exposed to Pb exhibit microglial activation, and upregulation of the level of IL-1b, TNF-a and iNOS, and these pro-inflammatory factors may cause hippocampal neuronal injury as well as Long Term Potentiation (LTP) deficits, These results suggest a direct link between Pb-induced neuroinflammation, neurodegeneration in hippocampus, and memory deficit (Liu et al., 2012). These effects are reversed by minocycline, an antibiotic which decreases microglial activation, strengthening the link between neuroinflammation, neurodegeneration and memory impairment. In epidemiological studies of adults, cumulative lifetime exposure to Pb has been associated with accelerated declines in cognition (Bakulski et al., 2012). In a study aiming at determining whether serum trace metals are related to abnormal cognition in Alzheimer's disease, it was found that serum Pb levels were significantly negatively correlated with verbal memory scores (Park et al., 2014). Cognitive impaiment was observed in mice exposed to Pb as infants but not as adults, suggesting that a window of vulnerability to Pb neurotoxicity can influence Alzheimer pathogenesis and cognitive decline in old age (Bihaqui et al., 2014). Human Tg-SWDI APP transgenic mice, which over-express amyloid plaques at age of 2-3 months, received oral gavage of 50 mg/kg of Pb once daily for 6 weeks. They showed a significant increase of Abeta in the CSF, brain cortex and hippocampus associated to impaired spatial learning ability, suggesting that Pb facilitates Abeta fibril formation and participate in deposition of amyloid plaques (Gu et al., 2012),</p>
<p>Wozniak et al., (2004) demonstrated that exposure of infant mice to EtOH on a single postnatal day (P7) induced extensive apoptotic neurodegeneration in the developing brain, and subsequent spatial learning and memory impairments that are very severe at P30, less severe if testing is first performed at P75, and minimal in later adulthood. In adulthood, working memory performance was also subtly compromised in EtOH-treated mice in a gender-dependent fashion, with the male EtOH mice being functionally impaired.</p>
<p>Allison et al., (2021) demonstrated that the deleterious effects of AD-related pathophysiology (i.e., higher levels of CSF ptau<sub>181</sub>/Aβ<sub>42</sub>) on verbal learning and memory performance depend on the degree of global atrophy present. More specifically, individuals with a greater degree of global atrophy evidenced similar rates of decline regardless of the degree of AD pathophysiology present. In contrast, in individuals with larger global brain volumes, the presence of preclinical Alzheimer's disease was associated with steeper declines in verbal learning and memory. These findings suggest that the presence of AD biomarkers, global atrophy, or both global atrophy and AD biomarkers are all associated with greater verbal learning and memory decline in a sample of late middle-aged adults.</p>
<p>Huang et al., (2012) findings indicated that exposure of P7 rats to ketamine leads to accelerated widespread neurodegeneration in the hippocampus. Suppression of p-PKCγ and p-ERK might be involved in neurologic damage, and this neurodegeneration could cause subsequent spatial learning abnormalities in adulthood.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>There are some inconsistencies regarding the time of exposure. Some papers clearly show that early Pb exposure increases amyloid and tau pathology and cognitive decline in aging. But few studies have addressed this complex question by using an ad hoc experimental design. Other studies have descibed the effects of lifetime or long-term exposure on cognitive functions but without a precise desciption of exposure onset and duration.</p>
<h4>References</h4>
<p>Allison, Samantha L., et al. "Neurodegeneration, Alzheimer's disease biomarkers, and longitudinal verbal learning and memory performance in late middle age." Neurobiology of aging 102 (2021): 151-160.</p>
<p>Bakulski KM, Park SK, Weisskopf MG, Tucker KL, Sparrow D, Spiro A, 3rd, et al. 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.</p>
<p>Barker GR, Warburton EC. 2011. When is the hippocampus involved in recognition memory? J Neurosci 31(29): 10721-10731.</p>
<p>Bihaqi SW, Bahmani A, Subaiea GM, Zawia NH. 2014. Infantile exposure to lead and late-age cognitive decline: relevance to AD. Alzheimer's & dementia : the journal of the Alzheimer's Association 10(2): 187-195.</p>
<p>Braskie MN, Thompson PM. 2013. Understanding cognitive deficits in Alzheimer's disease based on neuroimaging findings. Trends in cognitive sciences 17(10): 510-516.</p>
<p>Eslamizade MJ, Madjd Z, Rasoolijazi H, Saffarzadeh F, Pirhajati V, Aligholi H, et al. 2016. Impaired Memory and Evidence of Histopathology in CA1 Pyramidal Neurons through Injection of Abeta1-42 Peptides into the Frontal Cortices of Rat. Basic and clinical neuroscience 7(1): 31-41.</p>
<p>Gu 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 in vivo lead exposure. Toxicol Lett 213(2): 211-219.</p>
<p>Gu H, Wei X, Monnot AD, Fontanilla CV, Behl M, Farlow MR, et al. 2011. Lead exposure increases levels of beta-amyloid in the brain and CSF and inhibits LRP1 expression in APP transgenic mice. Neurosci Lett 490(1): 16-20.</p>
<p>Guerrero-Munoz MJ, Gerson J, Castillo-Carranza DL. 2015. Tau Oligomers: The Toxic Player at Synapses in Alzheimer's Disease. Frontiers in cellular neuroscience 9: 464.</p>
<p>Huang, Lining, et al. "Ketamine potentiates hippocampal neurodegeneration and persistent learning and memory impairment through the PKCγ–ERK signaling pathway in the developing brain." Brain research 1476 (2012): 164-171.</p>
<p>Liu MC, Liu XQ, Wang W, Shen XF, Che HL, Guo YY, et al. 2012. Involvement of microglia activation in the lead induced long-term potentiation impairment. PLoS One 7(8): e43924.</p>
<p>Park JH, Lee DW, Park KS, Joung H. 2014. Serum trace metal levels in Alzheimer's disease and normal control groups. American journal of Alzheimer's disease and other dementias 29(1): 76-83.</p>
<p>Salgado-Puga K, Pena-Ortega F. 2015. Cellular and network mechanisms underlying memory impairment induced by amyloid beta protein. Protein and peptide letters 22(4): 303-321.</p>
<p>Schneider JS, Anderson DW, Talsania K, Mettil W, Vadigepalli R. 2012. Effects of developmental lead exposure on the hippocampal transcriptome: influences of sex, developmental period, and lead exposure level. Toxicol Sci 129(1): 108-125.</p>
<p>Selkoe DJ, Hardy J. 2016. The amyloid hypothesis of Alzheimer's disease at 25 years. EMBO molecular medicine 8(6): 595-608.</p>
<p>Wozniak, David F., et al. "Apoptotic neurodegeneration induced by ethanol in neonatal mice is associated with profound learning/memory deficits in juveniles followed by progressive functional recovery in adults." Neurobiology of disease 17.3 (2004): 403-414.</p>
</div>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2968">Relationship: 2968: Increase, intracellular calcium leads to Apoptosis</a></h4>
<p>It is well established that variations in cytosolic calcium concentration [Ca<sup>2+</sup>]<sub>c</sub> trigger key cellular functions, for example, contraction of myofilaments, secretion of hormones and neurotransmitters and modulation of metabolism (Berridge et al., 2003; Rizzuto and Pozzan 2006; Clapham 2007). Moreover, Ca<sup>2+</sup> also has a major function in triggering mitotic division in numerous cell types (e.g., T lymphocytes and of oocytes) and, conversely, in the regulation of cell death (Giorgi et al., 2008). The notion that the cellular Ca<sup>2+</sup> overload is highly toxic, causing massive activation of proteases and phospholipases was known to cell biologists since the late 1960s (Pinton et al., 2008).</p>
<strong>Empirical Evidence</strong>
<div>
<p>Calcium is a ubiquitous intracellular signal responsible for controlling numerous cellular processes including cell proliferation, differentiation, and survival/death <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Clapham 2007)</span></span>. Studies have shown that Cd disrupts intracellular free calcium ([Ca<sup>2+</sup>]<sub>i</sub>) homeostasis, leading to apoptosis in a variety of cells, such as skin epidermal cells <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Son et al., 2010)</span></span>, hepatic cells <span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Lemarie et al., 2004; Xie et al., 2010)</span></span>, lymphoblastoid cells <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Lemarie et al., 2004)</span></span>, mesangial cells <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Wang et al., 2008; </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Liu and Templeton 2008; Yang et al., 2009)</span></span>, renal tubular cells <span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Yeh et al., 2009<sup>; </sup>Wang et al., 2009)</span></span>, astrocytes <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Yang et al., 2008)</span></span>, NIH 3T3 cells <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Biagioli et al., 2008)</span></span>, thyroid cancer cells <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Liu et al., 2007)</span></span>, and thymocytes <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Shen et al., 2001)</span></span>.</p>
<p>Baoshan et al. (2011) determined the role of calcium signaling in Cd-induced neuronal apoptosis. PC12 and SH-SY5Y cells, respectively, were treated with 0–20 µM Cd for 24 h, or with 10 and 20 µM Cd for 0–24 h. Subsequently, [Ca<sup>2+</sup>]<sub>i</sub> was measured with a calcium indicator dye, Fluo-3/AM or Fluo-4/AM. We found that treatment with Cd (0–20 µM) resulted in a concentration-dependent increase of [Ca<sup>2+</sup>]<sub>i</sub> in PC12 cells. Cd also induced a time-dependent elevation of [Ca<sup>2+</sup>]<sub>i</sub> in the cells during the period of 24 h. Similarly, Cd markedly elicited high [Ca<sup>2+</sup>]<sub>i</sub> fluorescence intensity in a concentration- and time-dependent manner in SH-SY5Y cells by fluorescence microscopy. Furthermore, Cd-elevated [Ca<sup>2+</sup>]<sub>i</sub> level was consistent with decreased cell viability or increased apoptosis of PC12 and SH-SY5Y cells <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Chen et al., 2008)</span></span>, suggesting that Cd-induced neuronal apoptosis might be associated with its induction of [Ca<sup>2+</sup>]<sub>i</sub> elevation <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Baoshan et al., 2011)</span></span>.</p>
<p>Yuan, Yan, et al. (2013) found that treatment with Cd (5, 10, 20 µM) resulted in a concentration-dependent increase of [Ca<sup>2+</sup>]<sub>i</sub> in cerebral cortical neurons. To verify the role of [Ca<sup>2+</sup>]<sub>i</sub> as a key second messenger, cells were pre-loaded with 10 µM BAPTA-AM for 30 min. Chelating intracellular Ca<sup>2+</sup> with BAPTA-AM prevented the elevation of [Ca<sup>2+</sup>]<sub>i</sub>, demonstrating that the release of intracellular Ca<sup>2+</sup> is essential for Cd-induced [Ca<sup>2+</sup>]<sub>i</sub> overloading. To explore other factors contributing to the calcium overload, we studied the effect of Cd on the activities of ATPases <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Yuan et al., 2013)</span></span>. Treatment of cerebral cortical neurons with Cd resulted in a significant loss in the activities of ATPases (<em>P</em><0.05 or <em>P</em><0.01), which occurred in a dose-dependent manner. When exposed to 5, 10 and 20 µM of Cd for 12 h, the Na<sup>+</sup>/K<sup>+</sup>-ATPase activity decreased to 70.1%, 52.5% and 27.2% of the control value while the Ca<sup>2+</sup>/Mg<sup>+</sup>-ATPase activity decreased to 62.6%, 49.0% and 25.5% of the control value, respectively. To examine the role of the ER in Cd-induced elevation of [Ca<sup>2+</sup>]<sub>i</sub>, we incubated neurons with 2-APB, a blocker of the ER calcium channel (inositol-1, 4, 5-trisphosphate receptor, IP<sub>3</sub>R). We observed that the elevation of [Ca<sup>2+</sup>]<sub>i</sub> induced by Cd was suppressed by 2-APB after treatment with Cd for 12 h. Taken together, these results demonstrated that [Ca<sup>2+</sup>]<sub>i</sub> elevation induced by Cd in cerebral cortical neurons is linked to the release of calcium from the ER <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Yuan et al., 2013)</span></span>. Next, to further determine the role of calcium in the regulation of Cd-induced apoptosis, cerebral cortical neurons were incubated with/without Cd (10 µM) in the absence or presence of BAPTA-AM (10 µM). Cd alone (10 µM) induced cell rounding and shrinkage, and BAPTA-AM itself did not alter cell shape. However, BAPTA-AM obviously blocked Cd-induced morphological changes. Furthermore, MTT assay results further demonstrated that BAPTA-AM in part can suppress Cd-induced loss of cell viability in Cd-exposed cerebral cortical neurons. These results suggest that Cd-induced neuronal apoptosis might be associated with its induction of [Ca<sup>2+</sup>]<sub>i</sub> elevation <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Yuan et al., 2013)</span></span>.</p>
<p>Independent evidence for the involvement of Ca<sup>2+</sup> influx in the triggering of apoptosis has come from studies with specific Ca<sup>2+</sup> channel blockers, which abrogate apoptosis in the regressing prostate following testosterone withdrawal (Martikainen and Isaacs 1990) and in pancreatic b-cells treated with serum from patients with type I diabetes (Juntti-Berggren et al., 1993). Other support for the involvement of Ca<sup>2+</sup> in apoptosis comes from the observation that agents which directly mobilize Ca<sup>2+</sup>can trigger apoptosis in diverse cell types (McConkey and Orrenius 1997).</p>
<p>Wyllie et al., (1984) demonstrated that Ca<sup>2+</sup> ionophores cause endonuclease activation as well as many of the morphological changes that are typical of apoptosis in thymocytes. Calcium ionophores also trigger apoptosis in prostate tumor cells (Martikainen and Isaacs 1990). Other support for this echanism has come from studies with the endoplasmic reticular Ca<sup>2+</sup>-ATPase inhibitor thapsigargin, the product of the plant, Thapsa garganica, which can also trigger all of the morphological and biochemical events of apoptosis in thymocytes (Jiang et al., 1994) and some other cell types (Levick et al., 1995; Kaneko and Tsukamoto 1994; Choi et al., 1995).</p>
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<strong>Uncertainties and Inconsistencies</strong>
<p>The duration and extent of Ca<sup>2+</sup> influx may determine whether cells survive, die by apoptosis, or undergo necrotic lysis (Choi 1995). According to this paradigm, continuous, but moderate increases in [Ca<sup>2+</sup>]<sub>i</sub> such as those produced by a sustained slow influx may cause apoptosis, whereas an exceedingly high influx rate would cause rapid cell lysis (Nicotera et al., 1998). For instance, collaborative work with Dr Stuart A. Lipton’s laboratory has shown that stimulation of cortical neurons with high concentrations of NMDA results in necrosis, whereas exposure to lower concentrations causes apoptosis (Bonfoco et al., 1995). Correspondingly, neuronal death in experimental stroke models is necrotic in the ischemic core, but delayed and apoptotic in the less severely compromised penumbra or border regions (Li et al., 1995; Charriaut-Marlangue et al., 1995). Further studies in our laboratories have shown that intracellular energy levels are rapidly dissipated in necrosis, but not in apoptosis (Cox et al., 1990; Matson et al., 1989). These results suggest that while initial events may be common to both types of cell death, certain metabolic conditions would be required to activate downstream controllers, which direct cells towards the organized execution of apoptosis (Leist and Nicotera 1997).</p>
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