<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Of the originating work: </span></span><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Jaeseong Jeong and Jinhee Choi, School of Environmental Engineering, University of Seoul, Seoul, Republic of Korea</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Of the content populated in the AOP-Wiki: </span></span><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">John R. Frisch and Travis Karschnik, General Dynamics Information Technology, Duluth, Minnesota; </span></span><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Daniel L. Villeneuve, US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, MN</span></span></p>
<td>Under development: Not open for comment. Do not cite</td>
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<div id="abstract">
<h2>Abstract</h2>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Reactive oxygen species (ROS) are derived from oxygen molecules and can occur as free radicals (ex. superoxide, hydroxyl, peroxyl) or non-radicals (ex. ozone, singlet oxygen). ROS production occurs via a variety of normal cellular process; however, in stress situations (ex. exposure to radiation, chemical or biological stressors) reactive oxygen species levels dramatically increase and cause damage to cellular components. In this Adverse Outcome Pathway (AOP) we focus on the inflammation response to increases in oxidative stress. Inflammation pathways include a molecular response (ex. interleukins, cytokines, interferons) and produces visible tissue swelling during histology examinations. In this AOP we focus on the apoptosis response to cellular damage. Pathways leading to apoptosis, or single cell death, have traditionally been studied as both independent and simultaneous from pathways leading to necrosis, or tissue-wide cell death, with both overlap and distinct mechanisms (Elmore 2007). For the purposes of this AOP, we are characterizing cancer due to widespread cell-death, and recognize the complications in separating the related apoptosis and necrosis pathways.</span></span></p>
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<div id="background">
<h3>Background</h3>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">This Adverse Outcome Pathway (AOP) focuses on the pathway in which an established molecular disruption, increased levels of reactive oxygen species (ROS), leads to increased cancer through inflammation and cell/death/apoptosis. Environmental stressors leading to increased reactive oxygen species result in a variety of stress responses, visible through inflammation. These stress responses have been studied in many eukaryotes, including mammals (humans, lab mice, lab rats), teleost fish, and invertebrates (cladocerans, mussels).</span></span></p>
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">This Adverse Outcome Pathway (AOP) focuses on the pathway in which an established molecular disruption, increased levels of reactive oxygen species (ROS), leads to increased cancer through inflammation and cell/death/apoptosis. Environmental stressors leading to increased reactive oxygen species result in a variety of stress responses, visible through inflammation. These stress responses have been studied in many eukaryotes, including mammals (humans, lab mice, lab rats), teleost fish, and invertebrates (cladocerans, mussels).</span></span></p>
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<div id="development_strategy">
<h3>Strategy</h3>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">This AOP was developed as part of an Environmental Protection Agency effort to represent putative AOPs from peer-reviewed literature which were heretofore unrepresented in the AOP-Wiki. Jeong and Choi (2020) and Jeong and Choi (2019) provided initial network analysis from microplastic stressors, guided by weight of evidence from ToxCast assays. These publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">The AOP-wiki authors did a further evaluation of published peer-reviewed literature to provide additional evidence in support of the AOP. A companion adverse outcome pathway is planned for an additional pathway initiated by reactive oxygen species (ROS), leading to increased cancer: Decreased, PPARalpha transactivation of gene expression leads to Alteration, lipid metabolism.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">1. Support for Biological Plausibility of Key Event Relationships: Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Level of Support</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#212529">Strong = Extensive understanding of the KER based on extensive previous documentation and broad acceptance.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:#212529">Strong support.</span></strong><span style="color:#212529"> The relationship between increases in reactive oxygen species and oxidative stress is broadly accepted and consistently supported across taxa.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:#212529">Strong support. </span></strong><span style="color:#212529">The relationship between increased inflammation and general apoptosis is established. Inflammation has been shown as an initiating event for activation of apoptosis; arguably more studies have been conducted linking inflammation to necrosis pathways.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:#212529">Strong support. </span></strong><span style="color:#212529">The relationship between failure of apoptosis pathways to initiate cell death pathways and increases in cancer is broadly accepted and consistently supported across taxa.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:#212529">Strong support. </span></strong><span style="color:#212529">Extensive understanding of the relationships between events from empirical studies from a variety of taxa.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Life Stage: The life stage applicable to this AOP is all life stages. Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles > embryos) due to accumulation of reactive oxygen species.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sex: This AOP applies to both males and females.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Taxonomic: This AOP appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats), teleost fish, and invertebrates (cladocerans, mussels).</span></span></p>
<h3>Essentiality of the Key Events</h3>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Support for the essentiality of the key events can be obtained from a wide diversity of taxonomic groups, with mammals (lab ice, lab rats, human cell lines), telost fish, and invertebrates (cladocerans and mussels) particularly well-studied.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">2. Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Level of Support</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Strong = Direct evidence from specifically designed experimental studies illustrating essentiality and direct relationship between key events.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Moderate = Indirect evidence from experimental studies inferring essentiality of relationship between key events due to difficulty in directly measuring at least one of key events.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> Increased Reactive oxygen species (ROS) levels are a primary cause of oxidative stress. Evidence is available from studies of stressor exposure and resulting changes in gene expression and protein/enzyme levels.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Oxidative stress is a cause of inflammation. Evidence is available from studies of stressor exposure and resulting changes in gene expression, protein/enzyme levels, and histology.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Inflammation is a cause of apoptosis. Evidence is available from studies of stressor exposure and resulting changes in gene expression, protein/enzyme levels, and histology.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Moderate support. </strong>Failure of apoptosis allows cancer cells to proliferate. Evidence is available from studies of stressor exposure and resulting changes in gene expression, protein/enzyme levels, and histology.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Cancer proliferates due to a variety of stressors and breakdown of multiple cellular processes. Evidence is available from studies of stressor exposure and resulting changes in gene expression, protein/enzyme levels, and histology.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Moderate to strong support. </strong>Direct evidence from empirical studies for most key events, with more inferential evidence rather than direct evidence for apoptosis.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Increased, Reactive oxygen species leads to Oxidative Stress</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Biological plausibility is high. Representative studies have been done with mammals (Liu et al. 2015; Deng et al. 2017; Schrinzi et al. 2017; Jeong and Choi 2020); fish (Oliveira et al. 2013; Lu et al. 2016; Alomar et al. 2017; Chen et al. 2017; Veneman et al. 2017; Barboza et al. 2018; Choi et al. 2018; Espinosa et al. 2018); invertebrates (Browne et al. 2013; Avio et al. 2015; Jeong et al. 2016, 2017; Paul-Pont et al. 2016; Imhof et al. 2017; Lei et al. 2018; Yu et al. 2018).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Biological plausibility is high. Representative studies have been done with mammals (Gamo et al. 2008; Jeong and Choi 2020); fish (Lu et al. 2016; Jin et al. 2018); invertebrates (Lei et al. 2018). For review (Wright and Kelly 2017).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Biological plausibility is high. Representative studies have been done with mammals (Gamo et al. 2008); fish (Karami et al. 2016; Lu et al. 2016; Jin et al. 2018). For review (Balkwill 2003, Villeneuve et al. 2018).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Biological plausibility is high. Representative studies have been done with mammals (Pavet et al. 2014; Jeong and Choi 2020). For review (Heinlein and Chang 2004; Vihervaara and Sistonen 2014).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3.<span style="background-color:#d0cece"> Empirical Support for Key Event Relationship: Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown?</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Level of Support</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Strong = Experimental evidence from exposure to toxicant shows consistent change in both events across taxa and study conditions.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> Increases in ROS lead to increases in oxidative stress, primarily from studies examining responses in enzyme and gene levels for enzymes that catalyze reactions that reduce ROS levels.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> Increases in oxidative stress leads to increases in inflammation, primarily from histology studies measuring tissue swelling, and increases in gene levels for proinflammatory mediators.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 2976: Increase, Inflammation leads to General Apoptosis</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> Increases in inflammation leads to apoptosis, primarily from studies of increased gene expression of tumor necrosis factor.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 2977: General Apoptosis leads to Increase, Cancer</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Mechanistic studies show that failure for apoptosis to eliminate cancer cells allows increases in cancer proliferation.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Exposure from empirical studies shows consistent change in both events from a variety of taxa</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Evidence from empirical studies shows consistent change in both events from a variety of taxa</span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">For overview of the biological mechanisms involved in this AOP, see Liu et al. (2015) and Jeong and Choi (2020); their studies analyzed ToxCast in vitro assays of mammalian acute toxicity data to identify correlations between toxicity pathways and chemical stressors, providing support for the key event relationships represented here.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Alomar, C., Sureda, A., Capo, X., Guijarro, B., Tejada, S. and Deudero, S. 2017. Microplastic ingestion by Mullus surmuletus Linnaeus, 1758 fish and its potential for causing oxidative stress. Environmental Research 159: 135-142.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Avio, C.G., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., D’Errico, G., Pauletto, M., Bargelloni, L., and Regoli, F. 2015. Pollutants bioavailability and toxicological risk from microplastics to marine mussels. Environmental Pollutants 198: 211-222.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Barboza, LG.A., Vieira, L.R., Branco, V., Figueiredo, N., Carvalho, F., Carvalho, C., and Guilhermino, L. 2018. Microplastics cause neurotoxicity, oxidative damage and energy-related changes and interact with the bioaccumulation of mercury in the European seabass, Dicentrachus labrux (Linneaeus, 1758). Aquatic Toxicology 195: 49-57.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Balkwill, F. 2003. Chemokine biology in cancer. Seminars in Immunology 15: 49-55.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Browne, M.A. Niven, S.J., Galloway, T.S., Rowland, S.J., and Thompson, R.C. 2013. Microplastic moves pollutants and additives to worms, reducing functions linked to health and biodiversity. Current Biology 23: 2388-2392.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Chen, Q., Gundlach, M., Yang, S., Jiang, J., Velki, M., Yin, D., and Hollert, H. 2017 Quantitative investigation of the mechanisms of microplastics and nanoplastics toward larvae locomotor activity. Science of the Total Environment 584-585: 1022-1031.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Choi, J.S., Jung, Y.J., Hong, N.H., Hong, S.H., and Park, J.W. 2018. Toxicological effects of irregularly shaped and spherical microplastics in a marine teleost, the sheepshead minnow (Cyprinodon variegatus). Marine Pollution Bulletin 129: 231-240.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Deng, Y., Zhang, Y., Lemos, B., and Ren, H. 2017. Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. Science Reports 7: 1-10.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Elmore, S. 2007. Apoptosis: A Review of Programmed Cell Death. Toxicologic pathology 35 (4): 495-516.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Espinosa, C., Garcia Beltran, J.M., Esteban, M.A., and Cuesta, A. 2018. In vitro effects of virgin microplastics on fish head-kidney leucocyte activities. Environmental Pollution 235: 30-38.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Gamo, K., Kiryu-Seo, S., Konishi, H., Aoki, S., Matushima, K., Wada, K., and Kiyama, H. 2008. G-protein-coupled receptor screen reveals a role for chemokine recepteor CCR5 in suppressing microglial neurotoxicity. Journal of Neuroscience 28: 11980-11988.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Heinlein, C.A. and Chang, C. 2004. Androgen receptor in prostate cancer. Endocrine Reviews 25: 276-308.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Imhof, H.K., Rusek, J., Thiel, M., Wolinska, J., and Laforsch, C. 2017. Do microplastic particles affect Daphnia magna at the morphological life history and molecular level? Public Library of Science One 12: 1-20.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Jeong, J. and Choi, J. 2019. Adverse outcome pathways potentially related to hazard identification of microplastics based on toxicity mechanisms. Chemosphere 231: 249-255.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Jeong, J. and Choi, J. 2020. Development of AOP relevant to microplastics based on toxicity mechanisms of chemical additives using ToxCast™ and deep learning models combined approach. Environment International 137:105557.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Jeong, C.B., Kang, H.M., Lee, M.C., Kim, D.H., Han, J., Hwang, D.S. Souissi, S., Lee, S.J., Shin, K.H., Park, H.G., and Lee, J.S. 2017. Adverse effects of microplastics and oxidative stress-induced MAPK/NRF2 pathway-mediated defense mechanisms in the marine copepod Paracyclopina nana. Science Reports 7: 1-11.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Jeong, C.B., Wong, E.J., Kang, H.M., Lee, M.C., Hwang, D.S., Hwang, U.K., Zhou, B., Souissi, S., Lee, S.J., and Lee, J.S. 2016. Microplastic size-dependent toxicity, oxidative stress induction, and p-JNK and p-p38 activation in the Monogonout rotifer (Brachionus koreanus). Environmental Science and Technology 50: 8849-8857.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Jin, Y., Xia, J., Pan, Z., Yang, J., Wang, W., and Fu, Z. 2018. Polystyrene microplastics induce microbiota dysbiosis and inflammation in the gut of adult zebrafish. Environmental Pollution 235: 322-329.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Karami, A., Romano, N., Galloway, T. and Hamzah, H. 2016. Virgin microplastics cause toxicity and modulate the impacts of phenanthrene on biomarker responses in African catfish (Clarias gariepinus). Environmental Research 151: 58-70.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Lei, L., Wu, S., Lu, S., Liu, M., Song, Y., Fu, Z., Shi, H., Raley-Susman, K.M., and He, D. 2018. Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematode Caenorhabditis elegans. Science of the Total Environment 619-620: 1-8.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Liu, J., Mansouri, K., Judson, R.S., Martin, M.T., Hong, H., Chen, M., Xu, X., Thomas, R.S., and Shah, I. 2015. Predicting hepatoxicity using ToxCast in vitro bioactivity and chemical structure. Chemical Research in Toxicology 28: 738-751.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Lu, Y., Zhang, Y., Dengy, Y., Jiang, W., Zhao, Y., Geng, J., Ding, L., Ren, H. 2016. Uptake and accumulation of polystyrene microplastics in zebrafish (Danio rerio) and toxic effects in liver. Environmental Science and Technology 50: 4054-4060.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Oliveira, M., Ribeiro, A., Hylland, K., and Guilhermino, L. 2013. Single and combined effects of microplastics and pyrene on juveniles (0+ group) of the common goby Pomatoschistus microps (Teleostei, Gobiidae). Ecological Indicators 34: 641-647.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Paul-Pont, I., Lacroix, C., Gonzalez Fernandez, D., Hegaret, H., Lambert, C., Le Goic, N., Frere, L., Cassone, A.L., Sussarellu, R. Fabioux, C., Guyomarch, J., Albentosa, M., Huvet, A., and Soudant, P. 2016. Exposure of marine mussels Mytillus spp. to polystyrene microplastics: Toxicity and influence on fluoranthene bioaccumulation. Environmental Pollution 216: 724-737.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Pavet, V., Shlyakhtina, Y., He, T., Ceschin, D.G., Kohonen, P., Perala, M., Kallioniemi, O., and Gronemeyer, H. 2014. Plasminogen activator urokinase expression reveals TRAIL responsiveness and support fractional survival of cancer cells. Cell Death and Disease 5: e1043.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Schrinzi, G.F., Perez-Pomeda, I., Sanchis, J., Rossini, C., Farre, M., and Barcelo, D. 2017. Cytotoxic effects of commonly used nanomaterials and microplastics on cerebral and epithelial human cells. Environmental Research 159: 579-587.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Veneman, W.J., Spaink, H.P., Brun, N.R., Bosker, T., and Vijver, M.G. 2017. Pathway analysis of systemic transcriptome responses to injected polystyrene particles in zebrafish larvae. Aquatic Toxicology 190: 112-120.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Vihervaara, A. and Sistonen, L. 2014. HSF1 at a glance. Journal of Cell Scientce 127: 261-266.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Villeneuve, D.L., Landesmann, B., Allavena, P., Ashley, N., Bal-Price, A., Corsini, E., Halappanavar, S., Hussell, T., Laskin, D., Lawrence, T., Nikolic-Paterson, D., Pallary, M., Paini, A., Pietrs, R., Roth, R., and Tschudi-Monnet, F. 2018. Toxicological Sciences 346:352.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Wright, S.L. and Kelly, F.J. 2017. Plastic and human health: a micro issue? Enviromental Science and Technology 51: 6634-6647.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Yu, P., Liu, Z., Wu, D., Chen, M., Lv, W., and Zhao, Y. 2018. Accumulation of polystyrene microplastics in juvenile Eriocheir sinensis and oxidative stress effects in the liver. Aquatic Toxicology 200: 28-36.</span></span></p>
<td><a href="/aops/383">Aop:383 - Inhibition of Angiotensin-converting enzyme 2 leading to liver fibrosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/382">Aop:382 - Angiotensin II type 1 receptor (AT1R) agonism leading to lung fibrosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/384">Aop:384 - Hyperactivation of ACE/Ang-II/AT1R axis leading to chronic kidney disease </a></td>
<td>KeyEvent</td>
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<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/409">Aop:409 - Frustrated phagocytosis leads to malignant mesothelioma</a></td>
<td>KeyEvent</td>
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<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>
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<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>
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<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>
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<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>
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<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>
<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>
</td>
</tr>
</tbody>
</table>
</div>
<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>
</div>
<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>
</div>
<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>
<p>Onoue S, Hosoi K, Wakuri S, Iwase Y, Yamamoto T, Matsuoka N, et al. Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation. Journal of applied toxicology : JAT. 2013;33:1241-50.</p>
<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>
<p>PCPC. PCPC 2014 safety evaluation guidelines; Chapter 7: Evaluation of Photoirritation and Photoallergy potential. Personal Care Products Council; 2014.</p>
<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>
<p>Seto Y, Kato M, Yamada S, Onoue S. Development of micellar reactive oxygen species assay for photosafety evaluation of poorly water-soluble chemicals. Toxicology in vitro : an international journal published in association with BIBRA. 2013;27:1838-46.</p>
<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/220">Aop:220 - Cyp2E1 Activation Leading to Liver Cancer</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>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/284">Aop:284 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress leads to chronic kidney disease</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<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>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/411">Aop:411 - Oxidative stress Leading to Decreased Lung Function </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/424">Aop:424 - Oxidative stress Leading to Decreased Lung Function via CFTR dysfunction</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/425">Aop:425 - Oxidative Stress Leading to Decreased Lung Function via Decreased FOXJ1</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/429">Aop:429 - A cholesterol/glucose dysmetabolism initiated Tau-driven AOP toward memory loss (AO) in sporadic Alzheimer's Disease with plausible MIE's plug-ins for environmental neurotoxicants</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/452">Aop:452 - Adverse outcome pathway of PM-induced respiratory toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/464">Aop:464 - Calcium overload in dopaminergic neurons of the substantia nigra leading to parkinsonian motor deficits</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/470">Aop:470 - Deposition of energy leads to vascular remodeling</a></td>
<td><a href="/aops/470">Aop:470 - Deposition of energy leads to abnormal vascular remodeling</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/478">Aop:478 - Deposition of energy leading to occurrence of cataracts</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<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>
<td>KeyEvent</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>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/482">Aop:482 - Deposition of energy leading to occurrence of bone loss</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/483">Aop:483 - Deposition of Energy Leading to Learning and Memory Impairment</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>KeyEvent</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/26">Aop:26 - Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/488">Aop:488 - Increased reactive oxygen species production leading to decreased cognitive function</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/437">Aop:437 - Inhibition of mitochondrial electron transport chain (ETC) complexes leading to kidney toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/535">Aop:535 - Binding and activation of GPER leading to learning and memory impairments</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/171">Aop:171 - Chronic cytotoxicity of the serous membrane leading to pleural/peritoneal mesotheliomas in the rat.</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/138">Aop:138 - Organic anion transporter (OAT1) inhibition leading to renal failure and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/177">Aop:177 - Cyclooxygenase 1 (COX1) inhibition leading to renal failure and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/186">Aop:186 - unknown MIE leading to renal failure and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/200">Aop:200 - Estrogen receptor activation leading to breast cancer </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/444">Aop:444 - Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<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>
<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>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/457">Aop:457 - Succinate dehydrogenase inhibition leading to increased insulin resistance through reduction in circulating thyroxine</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/459">Aop:459 - AhR activation in the thyroid leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/507">Aop:507 - Nrf2 inhibition leading to vascular disrupting effects via inflammation pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/509">Aop:509 - Nrf2 inhibition leading to vascular disrupting effects through activating apoptosis signal pathway and mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/510">Aop:510 - Demethylation of PPAR promotor leading to vascular disrupting effects</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>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/538">Aop:538 - Adverse outcome pathway of PFAS-induced vascular disrupting effects via activating oxidative stress related pathways </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/260">Aop:260 - CYP2E1 activation and formation of protein adducts leading to neurodegeneration</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/450">Aop:450 - Inhibition of AChE and activation of CYP2E1 leading to sensory axonal peripheral neuropathy and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/501">Aop:501 - Excessive iron accumulation leading to neurological disorders</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>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/471">Aop:471 - Various neuronal effects induced by elavl3, sox10, and mbp</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/31">Aop:31 - Oxidation of iron in hemoglobin leading to hematotoxicity</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/462">Aop:462 - Activation of reactive oxygen species leading the atherosclerosis</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>KeyEvent</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>KeyEvent</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>KeyEvent</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>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>KeyEvent</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><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>KeyEvent</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>KeyEvent</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>KeyEvent</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>KeyEvent</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>KeyEvent</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>KeyEvent</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>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<h3>Evidence for Perturbation by Stressor</h3>
<h4>Platinum</h4>
<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">Kruidering et al. (1997) examined the effect of platinum on pig kidneys and found that it was able to induce significant dose-dependant ROS formation within 20 minutes of treatment administration.</span></span></span></span></p>
</p>
<h4>Aluminum</h4>
<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">In a study of the effects of aluminum treatment on rat kidneys, Al Dera (2016) found that renal GSH, SOD, and GPx levels were significantly lower in the treated groups, while lipid peroxidation levels were significantly increased. </span></span></span></span></p>
</p>
<h4>Cadmium</h4>
<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">Belyaeva et al. (2012) investigated the effect of cadmium treatment on human kidney cells. They found that cadmium was the most toxic when the sample was treated with 500 μM for 3 hours (Belyaeva et al., 2012). As this study also looked at mercury, it is worth noting that mercury was more toxic than cadmium in both 30-minute and 3-hour exposures at low concentrations (10-100 μM) (Belyaeva et al., 2012). </span></span></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">Wang et al. (2009) conducted a study evaluating the effects of cadmium treatment on rats and found that the treated group showed a significant increase in lipid peroxidation. They also assessed the effects of lead in this study, and found that cadmium can achieve a very similar level of lipid peroxidation at a much lower concentration than lead can, implying that cadmium is a much more toxic metal to the kidney mitochondria than lead is (Wang et al., 2009). They also found that when lead and cadmium were applied together they had an additive effect in increasing lipid peroxidation content in the renal cortex of rats (Wang et al., 2009).</span></span></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">Jozefczak et al. (2015) treated <em>Arabidopsis thaliana </em>wildtype, <em>cad2-1</em> mutant, and <em>vtc1-1</em> mutant plants with cadmium to determine the effects of heavy metal exposure to plant mitochondria in the roots and leaves. They found that total GSH/GSG ratios were significantly increased after cadmium exposure in the leaves of all sample varieties and that GSH content was most significantly decreased for the wildtype plant roots (Jozefczak et al., 2015). </span></span></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">Andjelkovic et al. (2019) also found that renal lipid peroxidation was significantly increased in rats treated with 30 mg/kg of cadmium.</span></span></span></span></p>
</p>
<h4>Mercury</h4>
<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">Belyaeva et al. (2012) conducted a study which looked at the effects of mercury on human kidney cells, they found that mercury was the most toxic when the sample was treated with 100 μM for 30 minutes. </span></span></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">Buelna-Chontal et al. (2017) investigated the effects of mercury on rat kidneys and found that treated rats had higher lipid peroxidation content and reduced cytochrome c content in their kidneys. </span></span></span></span></p>
</p>
<h4>Uranium</h4>
<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">In Shaki et al.’s article (2012), they found rat kidney mitochondria treated with uranyl acetate caused increased formation of ROS, increased lipid peroxidation, and decreased GSH content when exposed to 100 μM or more for an hour.</span></span> </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">Hao et al. (2014),</span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"> found that human kidney proximal tubular cells (HK-2 cells) treated with uranyl nitrate for 24 hours with 500 μM showed a 3.5 times increase in ROS production compared to the control. They also found that GSH content was decreased by 50% of the control when the cells were treated with uranyl nitrate (Hao et al., 2014). </span></span></span></span></p>
</p>
<h4>Arsenic</h4>
<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">Bhadauria and Flora (2007) studied the effects of arsenic treatment on rat kidneys. They found that lipid peroxidation levels were increased by 1.5 times and the GSH/GSSG ratio was decreased significantly (Bhadauria and Flora, 2007). </span></span></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">Kharroubi et al. (2014) also investigated the effect of arsenic treatment on rat kidneys and found that lipid peroxidation was significantly increased, while GSH content was significantly decreased. </span></span></span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">In their study of the effects of arsenic treatment on rat kidneys, Turk et al. (2019) found that lipid peroxidation was significantly increased while GSH and GPx renal content were decreased.</span></span></p>
</p>
<h4>Silver </h4>
<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">Miyayama et al. (2013) investigated the effects of silver treatment on human bronchial epithelial cells and found that intracellular ROS generation was increased significantly in a dose-dependant manner when treated with 0.01 to 1.0 μM of silver nitrate.</span></span></span></span></p>
</p>
<h4>Manganese</h4>
<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">Chtourou et al. (2012</span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">) investigated the effects of manganese treatment on rat kidneys. They found that manganese treatment caused significant increases in ROS production, lipid peroxidation, urinary H<sub>2</sub>O<sub>2</sub> levels, and PCO production. They also found that intracellular GSH content was depleted in the treated group (Chtourou et al., 2012). </span></span></span></span></p>
</p>
<h4>Nickel</h4>
<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">Tyagi et al. (2011) conducted a study of the effects of nickel treatment on rat kidneys. They found that the treated rats showed a significant increase in kidney lipid peroxidation and a significant decrease in GSH content in the kidney tissue (Tyagi et al., 2011). </span></span></span></span></p>
</p>
<h4>Zinc</h4>
<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">Yeh et al. (2011) investigated the effects of zinc treatment on rat kidneys and found that treatment with 150 μM or more for 2 weeks or more caused a time- and dose-dependant increase in lipid peroxidation. They also found that renal GSH content was decreased in the rats treated with 150</span></span> <span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">μM or more for 8 weeks (Yeh et al., 2011). </span></span></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">It should be noted that Hao et al. (2014) found that rat kidneys exposed to lower concentrations of zinc (such as 100 μM) for short time periods (such as 1 day), showed a protective effect against toxicity induced by other heavy metals, including uranium. Soussi, Gargouri, and El Feki (2018) also found that pre-treatment with a low concentration of zinc (10 mg/kg treatment for 15 days) protected the renal cells of rats were from changes in varying oxidative stress markers, such as lipid peroxidation, protein carbonyl, and GPx levels. </span></span></span></span></p>
</p>
<h4>nanoparticles</h4>
<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"><span style="color:black">Huerta-García et al. (2014) conducted a study of the effects of titanium nanoparticles on human and rat brain cells. They found that both the human and rat cells showed time-dependant increases in ROS when treated with titanium nanoparticles for 2 to 6 hours (Huerta-García et al., 2014). They also found elevated lipid peroxidation that was induced by the titanium nanoparticle treatment of human and rat cell lines in a time-dependant manner (Huerta-García et al., 2014). </span></span></span></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"><span style="color:black">Liu et al. (2010) also investigated the effects of titanium nanoparticles, however they conducted their trials on rat kidney cells. They found that ROS production was significantly increased in a dose dependant manner when treated with 10 to 100 μg/mL of titanium nanoparticles (Liu et al., 2010).</span></span></span></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"><span style="color:black">Pan et al. (2009) treated human cervix carcinoma cells with gold nanoparticles (Au1.4MS) and found that intracellular ROS content in the treated cells increased in a time-dependant manner when treated with 100 μM for 6 to 48 hours. They also compared the treatment with Au1.4MS gold nanoparticles to treatment with Au15MS treatment, which are another size of gold nanoparticle (Pan et al., 2009). The Au15MS nanoparticles were much less toxic than the Au1.4MS gold nanoparticles, even when the Au15MS nanoparticles were applied at a concentration of 1000 μM (Pan et al., 2009). When investigating further markers of oxidative stress, Pan et al. (2009) found that GSH content was greatly decreased in cells treated with gold nanoparticles. </span></span></span></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"><span style="color:black">Ferreira et al. (2015) also studied the effects of gold nanoparticles. They exposed rat kidneys to GNPs-10 (10 nm particles) and GNPs-30 (30 nm particles), and found that lipid peroxidation and protein carbonyl content in the rat kidneys treated with GNPs-30 and GNPs-10, respectively, were significantly elevated. </span></span></span></span></span></p>
<p><span style="color:#27ae60"><strong>Taxonomic applicability: </strong>Occurrence of oxidative stress is not species specific. </span></p>
<p><span style="color:#27ae60"><strong>Life stage applicability:</strong> Occurrence of oxidative stress is not life stage specific. </span></p>
<p><span style="color:#27ae60"><strong>Sex applicability: </strong>Occurrence of oxidative stress is not sex specific. </span></p>
<p><span style="color:#27ae60"><strong>Evidence for perturbation by prototypic stressor:</strong> There is evidence of the increase of oxidative stress following perturbation from a variety of stressors including exposure to ionizing radiation and altered gravity (Bai et al., 2020; Ungvari et al., 2013; Zhang et al., 2009). </span></p>
<h4>Key Event Description</h4>
<p style="text-align:justify">Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell. As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al. 2009) and can be used as indicators of the presence of oxidative stress in the cell.</p>
<p>Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell. As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al., 2009) and can be used as indicators of the presence of oxidative stress in the cell. </p>
<p style="text-align:justify">In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides (2SH àSS) on neighboring amino acids (Antelmann and Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010).</p>
<p>In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides on neighboring amino acids (Antelmann & Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010). </p>
<p><span style="font-size:16px"><span style="background-color:white"><span style="color:#2f5597">ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase, <span style="background-color:white">catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol</span></span></span><span style="color:#2f5597">, or antioxidant vitamins and minerals including vitamin E, C, carotene, lutein, zeaxanthin, selenium, and zinc (Fletcher, 2010). The enzymes, vitamins and minerals catalyze the conversion of ROS to non-toxic molecules such as water and O<sub>2</sub></span><span style="background-color:white"><span style="color:#2f5597"><span style="background-color:white">. However, these antioxidant systems are not perfect and endogenous metabolic processes and/or exogenous oxidative influences can trigger cumulative oxidative injuries to the mitochondria, causing a decline in their functionality and efficiency, which further promotes cellular oxidative stress (</span></span></span></span><span style="color:#2f5597">Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017)<span style="font-size:16px"><span style="background-color:white"><span style="background-color:white">.</span></span></span></span></p>
<p>ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase,catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol, or antioxidant vitamins and minerals including vitamin E, C, carotene, lutein, zeaxanthin, selenium, and zinc (Fletcher, 2010). The enzymes, vitamins and minerals catalyze the conversion of ROS to non-toxic molecules such as water and O2. However, these antioxidant systems are not perfect and endogenous metabolic processes and/or exogenous oxidative influences can trigger cumulative oxidative injuries to the mitochondria, causing a decline in their functionality and efficiency, which further promotes cellular oxidative stress (Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017). </p>
<p><span style="color:#27ae60"><span style="font-size:18px"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif"><span style="background-color:white">However, an emerging viewpoint suggests that ROS-induced modifications may not be as detrimental as previously thought, but rather contribute to signaling processes (Foyer et al., 2017). </span></span></span></span></span></span></p>
<p>However, an emerging viewpoint suggests that ROS-induced modifications may not be as detrimental as previously thought, but rather contribute to signaling processes (Foyer et al., 2017). </p>
<p style="text-align:justify">Protection against oxidative stress is relevant for all tissues and organs, although some tissues may be more susceptible. For example, the brain possesses several key physiological features, such as high O2 utilization, high polyunsaturated fatty acids content, presence of autooxidable neurotransmitters, and low antioxidant defenses as compared to other organs, that make it highly susceptible to oxidative stress (Halliwell, 2006; Emerit and al., 2004; Frauenberger et al., 2016).</p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Sources of ROS Production</span></span></strong></span></span></p>
<p><strong>Sources of ROS Production </strong></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Direct Sources:</span></span></strong><span style="font-size:12.0pt"><span style="color:#2f5597"> Direct sources involve the deposition of energy onto water molecules, breaking them into active radical species. When ionizing radiation hits water, it breaks it into hydrogen (H*) and hydroxyl (OH*) radicals by destroying its bonds. The hydrogen will create hydroxyperoxyl free radicals (HO<sub>2</sub>*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and more O<sub>2</sub> (Elgazzar and Kazem, 2015). Antioxidant mechanisms are also affected by radiation, with catalase (CAT) and peroxidase (POD) levels rising as a result of exposure (Seen et al. 2018; Ahmad et al. 2021). </span></span></span></span></p>
<p><strong>Direct Sources:</strong>Direct sources involve the deposition of energy onto water molecules, breaking them into active radical species. When ionizing radiation hits water, it breaks it into hydrogen (H*) and hydroxyl (OH*) radicals by destroying its bonds. The hydrogen will create hydroxyperoxyl free radicals (HO2*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H2O2) and more O2 (Elgazzar and Kazem, 2015). Antioxidant mechanisms are also affected by radiation, with catalase (CAT) and peroxidase (POD) levels rising as a result of exposure (Seen et al. 2018; Ahmad et al. 2021). </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Indirect Sources:</span></span></strong><span style="font-size:12.0pt"><span style="color:#2f5597"> An indirect source of ROS is the mitochondria, which is one of the primary producers in eukaryotic cells (Powers et al., 2008). As much as 2% of the electrons that should be going through the electron transport chain in the mitochondria escape, allowing them an opportunity to interact with surrounding structures. Electron-oxygen reactions result in free radical production, including the formation of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O<sub>2</sub>, and inorganic phosphate (P<sub>i</sub>) (Hargreaves et al. 2020; Raimondi et al. 2020; Vargas-Mendoza et al. 2021). The first and third complexes of the transport chain are the most relevant to mammalian ROS production (Raimondi et al., 2020). The mitochondria have its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS are also produced through nicotinamide adenine dinucleotide phosphate oxidase (NOX) stimulation, an event commenced by angiotensin II, a product/effector of the renin-angiotensin system (Nguyen Dinh Cat et al. 2013; Forrester et al. 2018). Other ROS producers include xanthine oxidase, immune cells (macrophage, neutrophils, monocytes, and eosinophils), phospholipase A<sub>2</sub> (PLA<sub>2</sub>), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021).</span></span></span></span></p>
<p><strong>Indirect Sources</strong>: An indirect source of ROS is the mitochondria, which is one of the primary producers in eukaryotic cells (Powers et al., 2008). As much as 2% of the electrons that should be going through the electron transport chain in the mitochondria escape, allowing them an opportunity to interact with surrounding structures. Electron-oxygen reactions result in free radical production, including the formation of hydrogen peroxide (H2O2) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O2, and inorganic phosphate (Pi) (Hargreaves et al. 2020; Raimondi et al. 2020; Vargas-Mendoza et al. 2021). The first and third complexes of the transport chain are the most relevant to mammalian ROS production (Raimondi et al., 2020). The mitochondria has its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS is also produced through nicotinamide adenine dinucleotide phosphate oxidase (Nox) stimulation, an event commenced by angiotensin II, a product/effector of the renin-angiotensin system (Nguyen Dinh Cat et al. 2013; Forrester et al. 2018). Other ROS producers include xanthine oxidase, immune cells (macrophage, neutrophils, monocytes, and eosinophils), phospholipase A2 (PLA2), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021). </p>
<h4>How it is Measured or Detected</h4>
<p><strong>Oxidative Stress. Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage.</strong><span style="color:#27ae60"> Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed</span></p>
<p><strong>Oxidative Stress:</strong> Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage.Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed </p>
<ul>
<li>Detection of ROS by chemiluminescence <span style="font-size:12px">(<span style="font-family:arial,helvetica,sans-serif">https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683)</span></span></li>
<li>Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential.</li>
<li>Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html). </li>
<li>Detection of ROS by chemiluminescence (https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683) </li>
<li>Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential. </li>
<li>Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html). </li>
<li>TBARS. Oxidative damage to lipids can be measured by assaying for lipid peroxidation using TBARS (thiobarbituric acid reactive substances) using a commercially available kit. </li>
<li>8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or HPLC, described in Chepelev et al. (Chepelev, et al. 2015).</li>
<li>8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or HPLC, described in Chepelev et al. (Chepelev, et al. 2015). </li>
</ul>
<p><strong>Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include:</strong></p>
<p> </p>
<p><strong>Molecular Biology:</strong> Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include: </p>
<ul>
<li>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus</li>
<li>Western blot for increased Nrf2 protein levels</li>
<li>Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleus</li>
<li>qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences)</li>
<li>Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014)</li>
<li>OECD TG422D describes an ARE-Nrf2 Luciferase test method</li>
<li>In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation</li>
<li>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleusWestern blot for increased Nrf2 protein levels </li>
<li>Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleusqPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences) </li>
<li>Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014) </li>
<li>OECD TG422D describes an ARE-Nrf2 Luciferase test method </li>
</ul>
<p> </p>
<p>In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation.</p>
<td><strong>Assay Type & Measured Content</strong></td>
<td><strong>Description</strong></td>
<td><strong>Dose Range Studied</strong></td>
<td>
<p><strong>Assay Characteristics </strong><strong>(Length / Ease of use/Accuracy)</strong></p>
<p><strong>Assay Type & Measured Content </strong></p>
</td>
<td>
<p><strong>Description </strong></p>
</td>
<td>
<p><strong>Dose Range Studied </strong></p>
</td>
<td>
<p><strong>Assay Characteristics (Length/Ease of use/Accuracy) </strong></p>
</td>
</tr>
<tr>
<td>
<p><strong>ROS Formation in the Mitochondria assay</strong> (Shaki et al., 2012)</p>
<p>ROS </p>
<p>Formation in the Mitochondria assay (Shaki et al., 2012) </p>
</td>
<td>“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 μM) in respiration buffer containing (0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 μM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 μM) to mitochondria and was then incubated for 10 min. Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.”</td>
<td>0, 50, 100 and 200 μM of Uranyl Acetate</td>
<td>
<p>Long/ Easy</p>
<p>“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 µM) in respiration buffer containing (0.32 mM sucrose, 10mM Tris, 20 mM Mops, 50 µM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 µM) to mitochondria and was then incubated for 10 min.Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.” </p>
<p>High accuracy</p>
<p> </p>
</td>
<td>
<p>0, 50,100 and 200 µM of Uranyl Acetate </p>
<td>“GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as μg/mg protein.”</td>
<p>Mitochondrial Antioxidant Content Assay Measuring GSH content (Shaki et al., 2012) </p>
<p> </p>
</td>
<td>
<p>0, 50, 100, or 200 <em>μ</em>M Uranyl Acetate</p>
<p>“GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as µg/mg protein.” </p>
</td>
<td>
<p>0, 50, </p>
<p>100, or </p>
<p>200 µM </p>
<p>Uranyl Acetate </p>
</td>
<td>
<p> </p>
</td>
<td> </td>
</tr>
<tr>
<td>
<p><strong>H<sub>2</sub>O<sub>2</sub> Production Assay</strong> Measuring H<sub>2</sub>O<sub>2</sub> Production in isolated mitochondria</p>
(Heyno et al., 2008)</td>
<td>“Effect of CdCl<sub>2</sub> and antimycin A (AA) on H<sub>2</sub>O<sub>2</sub> production in isolated mitochondria from potato. H<sub>2</sub>O<sub>2</sub> production was measured as scopoletin oxidation. Mitochondria were incubated for 30 min in the measuring buffer(see the Materials and Methods) containing 0.5 mM succinate as an electron donor and 0.2 µM mesoxalonitrile 3‐chlorophenylhydrazone (CCCP) as an uncoupler, 10 U horseradish peroxidase and 5 µM scopoletin.” (</td>
<p>H2O2 Production Assay Measuring H2O2 Production in isolated mitochondria(Heyno et al., 2008) </p>
<p> </p>
</td>
<td>
<p>“Effect of CdCl2 and antimycin A (AA) on H2O2 production in isolated mitochondria from potato. H2O2 production was measured as scopoletin oxidation. Mitochondria were incubated for 30min in the measuring buffer </p>
<p>(see the Materials and Methods) containing 0.5mM succinate as an electron donor and 0.2µM mesoxalonitrile 3‐chlorophenylhydrazone (CCCP) as an uncoupler, 10U horseradish peroxidase and 5µM scopoletin.” </p>
<p><strong>Flow Cytometry ROS & Cell Viability</strong></p>
(Kruiderig et al., 1997)</td>
<td>“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At <em>t </em>5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”</td>
<td> </td>
<p>Flow Cytometry ROS & Cell Viability (Kruiderig et al., 1997) </p>
<p> </p>
</td>
<td>
<p>“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)” </p>
</td>
<td>
<p> </p>
</td>
<td>
<p>Strong/easy</p>
medium</td>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>Strong/easy medium </p>
</td>
</tr>
<tr>
<td>
<p><strong>DCFH-DAAssay</strong> Detection of hydrogen peroxide production (Yuan et al.,2016)</p>
<p>DCFH-DA </p>
<p>Assay Detection of hydrogen peroxide production (Yuan et al., </p>
<p>2016) </p>
</td>
<td>
<p>Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H<sub>2</sub>O<sub>2 </sub>to form fluorescent production. </p>
<p>Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H2O2 to form fluorescent production. </p>
<p> </p>
</td>
<td>0-400 µM</td>
<td>
<p>Long/ Easy</p>
<p>0-400 </p>
<p>High accuracy</p>
<p>µM </p>
</td>
<td>
<p>Long/ Easy High accuracy </p>
</td>
</tr>
<tr>
<td>
<p><strong>H2-DCF-DAAssay</strong> Detection of superoxide production (Thiebault etal., 2007)</p>
<p>H2-DCF-DAAssay Detection of superoxide production (Thiebault etal., 2007) </p>
<p> </p>
</td>
<td>
<p>This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer. </p>
</td>
<td>This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer.</td>
<td>0–600 µM</td>
<td>
<p>Long/ Easy</p>
<p>0–600 </p>
<p>High accuracy</p>
<p>µM </p>
</td>
<td>
<p>Long/ Easy High accuracy </p>
</td>
</tr>
<tr>
<td><strong>CM-H2DCFDAAssay</strong></td>
<td>**Come back and explain the flow cytometry determination of oxidative stress from Pan et al. (2009)**</td>
<p>The dye (CM-H2DCFDA) diffuses into the cell and is cleaved by esterases, the thiol reactive chlormethyl group reacts with intracellular glutathione which can be detected using flow cytometry. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Lu, C. et al., 2006; </span></span></span></span></p>
<td>
<p>(Lu, C. et al., 2006; </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">ROS can induce electron transitions in molecules, leading to electronically excited products. When the electrons transition back to ground state, chemiluminescence is emitted and can be measured. Reagents such as uminol and lucigenin are commonly used to amplify the signal. </span></span></span></span></p>
<td>
<p>ROS can induce electron transitions in molecules, leading to electronically excited products. When the electrons transition back to ground state, chemiluminescence is emitted and can be measured. Reagents such as luminoland lucigenin are commonly used to amplify the signal. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">NO has a short half-life. However, if it has been reduced to nitrite (NO2-), stable azocompounds can be formed via the Griess Reaction, and further measured by spectrophotometry. </span></span></span></span></p>
<td>
<p>NO has a short half-life. However, if it has been reduced to nitrite (NO2-), stableazocompoundscan be formed via the Griess Reaction, and further measured by spectrophotometry. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The unpaired electrons (free radicals) found in ROS can be detected with EPR, and is known as electron paramagnetic resonance. A variety of spin traps can be used. </span></span></span></span></p>
<td>
<p>The unpaired electrons (free radicals) found in ROS can be detected with EPR and is known as electron paramagnetic resonance. A variety of spin traps can be used. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The Nitroblue Tetrazolium assay is used to measure O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> levels. O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> reduces nitroblue tetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm. </span></span></span></span></p>
<td>
<p>The NitroblueTetrazolium assay is used to measure O2.− levels. O2.− reducesnitrobluetetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescence analysis of DHE is used to measure O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> levels. O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> is reduced to O2 as DHE is oxidized to 2-hydroxyethidium, and this reaction can be measured by fluorescence. Similarly, hydrocyans can be oxidized by any ROS, and measured via fluorescence. </span></span></span></span></p>
<td>
<p>Fluorescence analysis of DHE is used to measure O2.− levels. O2.− is reduced to O2 as DHE is oxidized to 2-hydroxyethidium, and this reaction can be measured by fluorescence. Similarly,hydrocyanscan be oxidized by any ROS, and measured via fluorescence. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescence analysis to measure extramitochondrial or extracellular H<sub>2</sub>O<sub>2</sub> levels. In the presence of horseradish peroxidase and H<sub>2</sub>O<sub>2</sub>, Amplex Red is oxidized to resorufin, a fluorescent molecule measurable by plate reader. </span></span></span></span></p>
<td>
<p>Fluorescence analysis to measure extramitochondrial or extracellular H2O2 levels. In the presence of horseradish peroxidase and H2O2, AmplexRed is oxidized to resorufin, a fluorescent molecule measurable by plate reader. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">An indirect fluorescence analysis to measure intracellular H<sub>2</sub>O<sub>2</sub> levels. H<sub>2</sub>O<sub>2</sub> interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it to dichlorofluorescein (DCF), a fluorescent product. </span></span></span></span></p>
<td>
<p>An indirect fluorescence analysis to measure intracellular H2O2 levels. H2O2 interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it todichlorofluorescein(DCF), a fluorescent product. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescent measurement of intracellular H<sub>2</sub>O<sub>2</sub> levels. HyPer is a genetically encoded fluorescent sensor that can be used for <em>in vivo</em> and<em> in situ </em>imaging. </span></span></span></span></p>
<td>
<p>Fluorescent measurement of intracellular H2O2 levels.HyPeris a genetically encoded fluorescent sensor that can be used forin vivo and in situimaging. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The cytochrome c reduction assay is used to measure O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> levels. O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> is reduced to O2 as ferricytochrome c is oxidized to ferrocytochrome c, and this reaction can be measured by an absorbance increase at 550 nm. </span></span></span></span></p>
<td>
<p>The cytochrome c reduction assay is used to measure O2.− levels. O O2.− is reduced to O2 as ferricytochrome c is oxidized toferrocytochromec, and this reaction can be measured by an absorbance increase at 550 nm. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The redox state of tissue is detected through nuclear magnetic resonance/magnetic resonance imaging, with the use of a nitroxide spin probe or biradical molecule. </span></span></span></span></p>
<td>
<p>The redox state of tissue is detected through nuclear magnetic resonance/magnetic resonance imaging, with the use of a nitroxide spin probe or biradical molecule. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Biesemann, N. et al., 2018) </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">A downstream target of the Nrf2 pathway is involved in GSH synthesis. As an indication of oxidation status, GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., </span></span><span style="color:#2f5597"><a href="http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html"><span style="font-size:12.0pt"><span style="color:#2f5597">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</span></span></a></span><span style="font-size:12.0pt"><span style="color:#2f5597">). </span></span></span></span></span></p>
<td>
<p>A downstream target of the Nrf2 pathway is involved in GSH synthesis. As an indication of oxidation status, GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g.,<a href="http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html" rel="noreferrer noopener" target="_blank">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</a>). </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit. </span></span></span></span></span></p>
<td>
<p>Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit. </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Can be determined with enzyme-linked immunosorbent assay (ELISA) or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress.</span></span></span></span></p>
<td>
<p>Can be determined with ELISA or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress. </p>
<p>The Seahorse XFp Analyzer provides information on mitochondrial function, oxidative stress, and metabolic dysfunction of viable cells by measuring respiration (oxygen consumption rate; OCR) and extracellular pH (extracellular acidification rate; ECAR). </p>
</td>
<td>
<p>No </p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Molecular Biology:</span></span></strong><span style="font-size:12.0pt"><span style="color:#2f5597"> Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include: </span></span></span></span></span></p>
<p> </p>
<p>Molecular Biology:Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include: </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Amsen, D., de Visser, K. E., and Town, T., 2009)</span></span></span></span></p>
<td>
<p>(Amsen, D., de Visser, K. E., and Town, T., 2009) </p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus </span></span></span></span></span></p>
<td>
<p>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus </p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences) </span></span></span></span></span></p>
<td>
<p>qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1,Gcl,Gst,Prx,TrxR,Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array fromSABiosciences) </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis</span></span></span></span></p>
<td>
<p>Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis </p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Jackson, A. F. et al., 2014)</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway</span></span></span></span></span></p>
<td>
<p>Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID,metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway </p>
<p style="margin-left:48px; text-align:left"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Ahmad, S. et al. (2021), “60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression”, <em>Journal Insect Science,</em> Vol. 21/5, Oxford University Press, Oxford, </span><a href="https://doi.org/10.1093/jisesa/ieab080" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/jisesa/ieab080</a></span></span></p>
<p>Ahmad, S. et al. (2021), “60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression”, Journal Insect Science, Vol. 21/5, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jisesa/ieab080" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jisesa/ieab080</a> </p>
<p style="margin-left:48px; text-align:left"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, <em>Antioxidants & Redox Signaling</em>, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3400" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1089/ars.2010.3400</a></span></span></p>
<p>Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, Antioxidants & Redox Signaling, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3400" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2010.3400</a> </p>
<p style="margin-left:48px"><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">Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in <em>Inflammation and Cancer</em>, Humana Press, Totowa, </span></span><a href="https://doi.org/10.1007/978-1-59745-447-6_5" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#1155cc">https://doi.org/10.1007/978-1-59745-447-6_5</span></span></span></a> </span></span></p>
<p>Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, <a href="https://doi.org/10.1007/978-1-59745-447-6_5" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/978-1-59745-447-6_5</a> </p>
<p style="margin-left:48px"><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"><span style="color:black">Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, <em>Journal of Proteome Research</em>, Vol. 14/2, American Chemical Society, Washington, </span></span></span><a href="https://doi.org/10.1021/pr501141b" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1021/pr501141b</span></span></a></span></span></p>
<p>Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, <a href="https://doi.org/10.1021/pr501141b" rel="noreferrer noopener" target="_blank">https://doi.org/10.1021/pr501141b</a> </p>
<p style="margin-left:48px"><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"><span style="color:black">Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, <em>International Journal of Radiation Biology</em>, Vol. 93/9, Informa, London, </span></span></span><a href="https://doi.org/10.1080/09553002.2017.1339332" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1080/09553002.2017.1339332</span></span></a></span></span></p>
<p>Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, <a href="https://doi.org/10.1080/09553002.2017.1339332" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2017.1339332</a> </p>
<p style="margin-left:48px"><span style="color:#27ae60">Azzam, E. I. et al. (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, Cancer Letters, Vol. 327/1-2, Elsevier, Ireland, https://doi.org/10.1016/j.canlet.2011.12.012 </span></p>
<p>Azzam, E. I. et al. (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, Cancer Letters, Vol. 327/1-2, Elsevier, Ireland, https://doi.org/10.1016/j.canlet.2011.12.012 </p>
<p style="margin-left:48px"><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"><span style="color:black">Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, <em>American Journal of Physiology - Cell Physiology</em>, Vol. 318/5, American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/ajpcell.00520.2019." style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/ajpcell.00520.2019.</span></span></a></span></span></p>
<p>Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, American Journal of Physiology - Cell Physiology, Vol. 318/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/ajpcell.00520.2019." rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/ajpcell.00520.2019.</a> </p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, <em>Journal of ocular pharmacology and therapeutics</em>, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, <a href="https://doi.org/10.1089/jop.2000.16.285.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a> </span></p>
<p>Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, Journal of ocular pharmacology and therapeutics, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, <a href="https://doi.org/10.1089/jop.2000.16.285.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a> </p>
<p style="margin-left:48px"><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">Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”, <em>Scientific Reports, </em>Vol. 8/1,</span></span> <span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Nature Portfolio, London, </span></span><a href="https://doi.org/10.1038/s41598-018-27614-8" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1038/s41598-018-27614-8</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p>Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”,Scientific Reports, Vol. 8/1, Nature Portfolio, London,<a href="https://doi.org/10.1038/s41598-018-27614-8" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41598-018-27614-8</a>. </p>
<p style="margin-left:48px"><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"><span style="color:black">Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in <em>The Pathophysiologic Basis of Nuclear Medicine</em>, Springer, New York, pp. 540-548</span></span></span></span></span></p>
<p>Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in The Pathophysiologic Basis of Nuclear Medicine, Springer, New York, pp. 540-548 </p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, <em>Ophthalmic Research</em>, Vol. 44, Karger International, Basel, <a href="https://doi.org/10.1159/000316476.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a> </span></p>
<p>Eruslanov, E., & Kusmartsev, S. (2010). Identification of ROS using oxidized DCFDA and flow-cytometry. Methods in molecular biology ,N.J., Vol. 594, https://doi.org/10.1007/978-1-60761-411-1_4 </p>
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<p>Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, Ophthalmic Research, Vol. 44, Karger International, Basel, <a href="https://doi.org/10.1159/000316476.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a> </p>
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<p style="margin-left:48px"><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"><span style="color:#212121">Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, <em>Journal of applied physiology</em>, Vol. 106/1, American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/japplphysiol.01278.2007" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/japplphysiol.01278.2007</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">.</span></span></span></span></span></p>
<p>Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, Journal of applied physiology, Vol. 106/1, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.01278.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/japplphysiol.01278.2007</a>. </p>
<p style="margin-left:48px"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Zhao, R. Z. et al. (2019), “Mitochondrial electron transport chain, ROS generation and uncoupling”, <em>International journal of molecular medicine</em>, Vol. 44/1, </span><span style="color:black">Spandidos</span><span style="background-color:white"><span style="color:black"> Publishing Ltd</span></span><span style="color:black">., Athens, </span><a href="https://doi.org/10.3892/ijmm.2019.4188" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3892/ijmm.2019.4188</a></span></span></p>
<p>Zhao, R. Z. et al. (2019), “Mitochondrial electron transport chain, ROS generation and uncoupling”, International journal of molecular medicine, Vol. 44/1, Spandidos Publishing Ltd., Athens, <a href="https://doi.org/10.3892/ijmm.2019.4188" rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/ijmm.2019.4188</a> </p>
<p><span style="font-size:16px"><span style="font-family:"Calibri",sans-serif">Taxonomic: appears to be present broadly, with representative studies focused on mammals (humans, lab mice, lab rats).</span></span></p>
<p> </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">Extensive data exists on the presence of inflammation in human</span><span style="font-family:"Times New Roman",serif"> (Coussens, Aggarwal, Hannhan, Mantovani..) In human, many examples of chronic inflammation leading to cancer or cancer progression exist. For instance, Helicobacter pylori infection leads to gut cancer (Wang).</span></span></span></p>
<p> </p>
<h4>Key Event Description</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Inflammation is complex to define. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Villeneuve et al. (2018) analyzed the varied biological responses, provided guidance to simplify the process representing inflammation in adverse outcome pathways, and recommended 3 key steps: 1. Tissue resident cell activation 2. Increased Pro-inflammatory mediators 3. Leukocyte recruitment/activation. Tissue resident cell activation generally occurs when healthy tissue is exposed to a stressor, or when damage occurs, initiating a signal response of pro-inflammatory mediators (ex. cytokines). Pro-inflammatory mediators result in the production of lipids and proteins, signaling, and initiate leukocyte recruitment/activation. Leukocyte recruitment/activation initiate inflammation and other morphological changes. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">In cancer, inflammation is a cascade of events created by the host in response to the spread of the cancer (Coussens and Werb, 2002). In response to an injury or the presence of cancer, the host heals itself through inflammation. Indeed, the activation and the migration of leukocytes (neutrophils, monocytes and eosinophils) to the wound induces the healing process. These inflammatory cells provide an extracellular matrix that forms upon which fibroblast and endothelial cells proliferate and migrate in order to recreate a normal environment. Damage to the epithelial layer initiate inflammatory reactions (Palmer et al. 2011). In cancer, this inflammatory state induces cell proliferation, increases the production of reactive oxygen species leading to oxidative DNA damage, and reduces DNA repair (Coussens and Werb, 2002). For review of inflammation caused by microplastics in mammals, see Wright and Kelly (2017).</span></span></p>
<p> </p>
<p> </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">Inflammation can be defined as the response of the organism to a tissue injury (Coussens). Indeed, in order to heal this injury, a multitude of chemical signals initiate and maintain a host response. Leukocytes </span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">(neutrophils, monocytes and eosinophils)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121"> are recruited to the site of the damage through the attraction by chemokines (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">TNF-α (tumour necrosis factor-α)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">, interleukines…). A </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">provisional extracellular matrix (ECM)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121"> is created, and f</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">ibroblast and endothelial cells proliferate and migrate</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121"> to it. Wound healing is an example of physiological inflammation and is self-limiting (Coussens). In case of a dysregulation, inflammation can lead to pathologies. </span></span></span></span></span><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:#212121">Inflammation can be </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">caused by physical injury, ischemic injury, infection, exposure to toxins, or other types of trauma</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121"> (Singh).</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">Inflammation was described as one of the hallmarks of cancer by Hannahan et al. as a response to tumor invasion through mainly two mechanisms: promoting genetic instatbility and supply pro-tumorogenic factors.</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">First, inflammation in cancer promotes genetic instability (</span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#303030">Mantovani</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#303030">, colotta). Macrophages, in contact with the inflammatory site can be responsible of a reactive stress oxygen reaction (ROS) (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#303030">Maeda</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#303030">, Pollard, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Grivennikov</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#303030">). Indeed, they </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">generate high levels of reactive oxygen and nitrogen species </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">which </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">produce mutagenic agents</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121"> (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">peroxynitrite</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">, which </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">in turn </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">cause</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">s</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">DNA </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">mutation</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">s.</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">Second, </span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">in inflammation, the tumor micro environment plays a critical role (Coussens). Indeed, in can supply </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#505050">growth factors, survival factors, proangiogenic factors, extracellular matrix-modifying enzymes that facilitate angiogenesis, invasion, and metastasis, and inductive signals that lead to activation of EMT and other hallmark-facilitating programs</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#505050"> (Hannahan). For example, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">macrophages can become tumor associated macrophage which promote </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">cell proliferation, angiogenesis, and invasio</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">n (Singh, Lin, Qian)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">.</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">Moreover, chronic inflammation can also lead to tumorigenesis (Karin, </span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Singh</span></span></span><span style="font-family:"Times New Roman",serif">). Indeed, since 1863, Virchow has hypothesized that chronic inflammation causes cell proliferation (Balkwill). </span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">According to Aggarwall, s</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">everal pro-inflammatory </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">markers</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">such as </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">TNF and members of its superfamily, IL-1alpha, IL-1beta, IL-6, IL-8, IL-18, chemokines, MMP-9, VEGF, COX-2, and 5-LOX</span></span></span><span style="font-family:"Times New Roman",serif"> <span style="background-color:white"><span style="color:#212121">mediate</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121"> suppression of apoptosis, proliferation, angiogenesis, invasion, and metastasis</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121"> (Aggarwal).</span></span></span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Inflammation is generally detected in histopathological examination of organs (ex. liver, intestines) or in changes in gene expression (ex. interleukins). Activation of the innate immune response and the release of various inflammatory cytokines can also be assessed (Flake and Morgan, 2017). </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:#303030">Several assays can be used to measure inflammation: </span></span></span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Histopathology on samples. Several scoring tools exist (Goeboes)</span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measuring chemokines in the blood (</span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">ELISA, multiplex bead assays</span></span></span> <span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">: </span></span></span><span style="font-family:"Times New Roman",serif">interleukines (IL1, IL6), TNF, interferon… ) (Brenner) and histopathology samples</span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measuring </span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Prostaglandin</span></span></span> <span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">levels, </span></span></span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">COX-2</span></span></span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121"> (</span></span></span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">ELISA</span></span></span><br />
<span style="font-family:"Times New Roman",serif"><span style="color:#212121"><span style="background-color:#fffcf0">Liquid chromatography/tandem mass spectrometry</span></span></span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">, IHC)</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Transcription factors : </span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">STAT3 Activation</span></span></span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">, </span></span></span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">NF-κB Activation</span></span></span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121"> (</span></span></span><span style="background-color:#fffcf0"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">ELISA</span></span></span><br />
<span style="font-family:"Times New Roman",serif"><span style="color:#212121"><span style="background-color:#fffcf0">RtPCR to measure mRNA</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Biomarkers (white cell count, CRP) ratios, and predictive score using </span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measuring ROS(</span><span style="background-color:#d3e3fd"><span style="font-family:"Times New Roman",serif"><span style="color:#040c28">DCFDA</span></span></span><span style="background-color:#d3e3fd"><span style="font-family:"Times New Roman",serif"><span style="color:#040c28">, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#222222">horseradish peroxidase (HRP)-oxidizing substrates</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#222222">, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#222222">SOD-inhibitable reduction of cytochrome c</span></span></span><span style="background-color:#d3e3fd"><span style="font-family:"Times New Roman",serif"><span style="color:#040c28">) (Murphy).</span></span></span></span></span></li>
<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">Methods are extensively reviewed in Marchand et al and Murphy et al.</span></span></span></p>
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<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:#333333">Lin, Y., Xu, J. & Lan, H. Tumor-associated macrophages in tumor metastasis: biological roles and clinical therapeutic applications. </span></span></span><em>J Hematol Oncol</em> <strong>12</strong>, 76 (2019). <a href="https://doi.org/10.1186/s13045-019-0760-3" style="color:#467886; text-decoration:underline">https://doi.org/10.1186/s13045-019-0760-3</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:#212121">Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010 Mar 19;140(6):883-99. doi: 10.1016/j.cell.2010.01.025. PMID: 20303878; PMCID: PMC2866629.</span></span></span></span></span></p>
<p style="text-align:justify"> </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:#222222">Murphy, M.P., Bayir, H., Belousov, V. </span></span></span><em>et al.</em> Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. <em>Nat Metab</em> <strong>4</strong>, 651–662 (2022). <a href="https://doi.org/10.1038/s42255-022-00591-z" style="color:#467886; text-decoration:underline">https://doi.org/10.1038/s42255-022-00591-z</a></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:#333333">Geboes K, Riddell R, Öst A<em>, et al</em></span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:#333333">A reproducible grading scale for histological assessment of inflammation in ulcerative colitis</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:Aptos,sans-serif"><em><span style="font-family:"Times New Roman",serif"><span style="color:#333333">Gut </span></span></em><span style="font-family:"Times New Roman",serif"><span style="color:#333333">2000;<strong>47:</strong>404-409.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Brenner DR, Scherer D, Muir K, Schildkraut J, Boffetta P, Spitz MR, Le Marchand L, Chan AT, Goode EL, Ulrich CM, Hung RJ. A review of the application of inflammatory biomarkers in epidemiologic cancer research. Cancer Epidemiol Biomarkers Prev. 2014 Sep;23(9):1729-51. doi: 10.1158/1055-9965.EPI-14-0064. Epub 2014 Jun 24. PMID: 24962838; PMCID: PMC4155060.</span></span></span></span></span></span></p>
<h4><a href="/events/1513">Event: 1513: General Apoptosis</a></h4>
<p>Taxonomic: appears to be present broadly among multicellular organisms.</p>
<h4>Key Event Description</h4>
<p><span style="font-size:16px"><span style="font-family:calibri,sans-serif">Apoptosis is the programmed cell death in general. This process is well regulated with a sequence of events before cell fragmentation occurs. Changes in the nucleus of a cell are the first step in apoptosis. Before that, other factors such as stress, inflammation, cell damage can induce expression or activation of signal proteins which will activate the pathway for apoptosis. Examples of proteins which are involved in apoptosis are the proteins p53, Bcl-2, JNK, and several caspases. When the first step is taken in the apoptosis process the cell will end in membrane-bounded apoptotic bodies. These bodies are cleared by macrophages or other cells where the degradation process starts within heteorphagosomes.</span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:16px">There are several possibilities to measure and detect apoptosis, some common techniques are: </span></p>
<ul>
<li><span style="font-size:16px">The detection of </span>Lactate dehydrogenase (<span style="font-size:16px">LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) substances which are released from cells which undergo apoptosis. </span></li>
<li><span style="font-size:16px">An older but effective technique it the annexin V – affinity assay. The principle of this assay is the high affinity binding between annexin V and phosphatidylserine. In a vital cell there is a membrane lipid asymmetry where phosphatidylserine molecules are facing the cytosol. During apoptosis the membrane lipid asymmetry is lost, and the phosphatidylserine molecules are expressed in the outer membrane. When annexin-V is present in combination with Ca<sup>2+</sup> it binds with high affinity to phosphatidylserine. With a hapten label at the annexin-V this process can be detected.</span></li>
<li><span style="font-size:16px"><span style="font-family:calibri,sans-serif">Another technique is the detection of cleaved caspase-3, which could be done with western blot or enzyme-linked immunosorbent assays. </span></span></li>
<li><span style="font-size:16px"><span style="font-family:calibri,sans-serif">Cytochrome c is also a protein which is released in an early stage of apoptosis. Detection of cytochrome c can be done with metal nanoclusters which have a fluorescent probe in addition to western blot assay.</span></span></li>
</ul>
<h4>References</h4>
<p>Shtilbans, V., Wu, M. & Burstein, D. E. Evaluation of apoptosis in cytologic specimens. <em>Diagnostic Cytopathology</em> <strong>38,</strong> 685–697 (2010).</p>
<p>Wu, J., Sun, J. & Xue, Y. Involvement of JNK and P53 activation in G2/M cell cycle arrest and apoptosis induced by titanium dioxide nanoparticles in neuron cells. <em>Toxicol. Lett.</em> <strong>199,</strong> 269–276 (2010).</p>
<p>Redza-Dutordoir, M. & Averill-Bates, D. A. Activation of apoptosis signalling pathways by reactive oxygen species. <em>Biochim. Biophys. Acta - Mol. Cell Res.</em> <strong>1863,</strong> 2977–2992 (2016).</p>
<p>Lossi, L., Castagna, C. & Merighi, A. Neuronal cell death: An overview of its different forms in central and peripheral neurons. in <em>Neuronal Cell Death: Methods and Protocols</em> 1–18 (2014). doi:10.1007/978-1-4939-2152-2_1</p>
<p>Van Engeland, M., Nieland, L. J. W., Ramaekers, F. C. S., Schutte, B. & Reutelingsperger, C. P. M. Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. <em>Cytometry</em> <strong>31,</strong> 1–9 (1998).</p>
<p>Shamsipur, M., Molaabasi, F., Hosseinkhani, S. & Rahmati, F. Detection of Early Stage Apoptotic Cells Based on Label-Free Cytochrome c Assay Using Bioconjugated Metal Nanoclusters as Fluorescent Probes. <em>Anal. Chem.</em> <strong>88,</strong> 2188–2197 (2016).</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Life Stage:</span> All life stages. Older individuals are more likely to manifest this key event (adults > juveniles > embryos).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Sex: A</span>pplies to both males and females.</span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">Taxonomic:</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> Appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats), teleost fish, and invertebrates (cladocerans, mussels).</span></span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Cancer is a general key event for related diseases each exhibiting uncontrolled proliferation of abnormal cells (for review see Hanahan and Weinberg 2011). A cancer often is initially associated with a specific organ, with malignant tumors developing ability to metastasize, or travel to other areas of the body. Most cancers develop from genetic mutations in normal cells, although a minority of cancers are hereditary. Exposure to chemical stressors, radiation, tobacco smoke, or viruses can increase the likelihood that cancer will develop.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Cancer cells proliferate due to capabilities summarized by Hanahan and Weinberg (2011): </span></span></p>
<ol>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sustained proliferation signaling – by deregulating normal cell signals, cancer cells can sustain chronic proliferation.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Evading growth suppressors – by evading activities of tumor suppressor genes, cancer cells continue to proliferate.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Activating invasion and metastasis – by altering shape and attachment to cells in the extracellular matrix, cancer cells gain ability to move to other locations.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Enabling replicative immortality – by disabling senescence pathways, cancer cells have extended lifespans.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Inducing angiogenesis – by enabling neovasculature, cancer cells receive nutrients and oxygen and get rid of waste products.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Resisting cell death – by evading apotosis and necrosis defense pathways, cancer cells avoid elimination.</span></span></li>
</ol>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Most carcinogenicity studies are conducted with rodents (see OECD 2018; Zhou et al. 2023 for methods) or in-vitro with mammalian cell lines (see OECD 2023 for methods). Cancer is usually detected by biopsy or histopathological examination of tissue. Gene expression levels can also be assessed, as increased transcription of known genes have been associated with specific cancers (ex. Tumor Necrosis Factor (Pavet et al. 2014); Heat Shock Factors (Vihervaara and Sistonen 2014; Androgen Receptor (Heinlein and Chang 2004)).</span></span></p>
<h4>Regulatory Significance of the AO</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Cancer is a critical endpoint in human health risk assessment. It is embedded in regulatory frameworks for human health protection in many countries (see OSHA 2023 for examples of US regulations and European Parliament 2022 for examples of regulations in Europe).</span></span></p>
<h4>References</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Abraha, A.M. and Ketema, E.B. 2016. Apoptotic pathways as a therapeutic target for colorectal cancer treatment. World Journal of Gastrointestinal Oncology 8 (8): 583-491</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">European Parliament. 2022. Directive 2004/37/EC of the European Parliament on the protection of workers from the risks related to exposure to carcinogens, mutagens or reprotoxic substances at work. Retrieved 3 August 2023 from http://data.europa.eu/eli/dir/2004/37/2022-04-05</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Hanahan, D. and Weinberg, R.A. 2011. Hallmarks of cancer: the next generation. Cell 144(5): 646-674.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Heinlein, C.A. and Chang, C. 2004. Androgen receptor in prostate cancer. Endocrine Reviews 25: 276-308.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">OECD. 2018. Test no. 451: OECD Guideline for the Testing of Chemicals: Carcinogenicity Studies. OECD Publishing, Paris. Retrieved 3 August 2023 from <a href="https://www.oecd.org/env/test-no-451-carcinogenicity-studies-9789264071186-en.htm" style="color:#0563c1; text-decoration:underline">https://www.oecd.org/env/test-no-451-carcinogenicity-studies-9789264071186-en.htm</a></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">OECD. 2023. Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. Retrieved 3 August 2023 from <a href="https://doi.org/10.1787/9789264264861-en.htm" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1787/9789264264861-en.htm</a></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">OSHA. 2023. Carcinogens. Retrieved 3 August 2023 from <a href="https://www.osha.gov/carcinogens/standards" style="color:#0563c1; text-decoration:underline">https://www.osha.gov/carcinogens/standards</a></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Pavet, V., Shlyakhtina, Y., He, T., Ceschin, D.G., Kohonen, P., Perala, M., Kallioniemi, O., and Gronemeyer, H. 2014. Plasminogen activator urokinase expression reveals TRAIL responsiveness and support fractional survival of cancer cells. Cell Death and Disease 5: e1043.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Vihervaara, A. and Sistonen, L. 2014. HSF1 at a glance. Journal of Cell Scientce 127: 261-266.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Zhou, Y., Xia, J., Xu, S., She, T., Zhang, Y., Sun, Y., Wen, M., Jiang, T., Xiong, Y., and Lei, J. 2023. Experimental mouse models for translational human cancer research. Frontiers in Immunology 14: 1095388.</span></span></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/2009">Relationship: 2009: Increased, Reactive oxygen species leads to Oxidative Stress </a></h4>
<h4><a href="/relationships/2009">Relationship: 2009: Increase, ROS leads to Oxidative Stress </a></h4>
<td><a href="/aops/505">Reactive Oxygen Species (ROS) formation leads to cancer via inflammation pathway</a></td>
<td>adjacent</td>
<td>High</td>
<td>Low</td>
<td>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/521">Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/186">unknown MIE leading to renal failure and mortality</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/497">ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/540">Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>adjacent</td>
<td>High</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/aops/462">Activation of reactive oxygen species leading the atherosclerosis</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/396">Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/26">Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/534">Succinate dehydrogenase (SDH) inhibition leads to cancer through oxidative stress</a></td>
<td><a href="/aops/481">AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/324">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/325">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/326">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/331">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/332">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/333">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/596">Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/599">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/600">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/601">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/602">Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/603">Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Life Stage: The life stage applicable to this key event relationship is all life stages. Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles > embryos) due to accumulation of reactive oxygen species.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sex: This key event relationship applies to both males and females.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Taxonomic: This key event relationship appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats), teleost fish, and invertebrates (cladocerans, mussels).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Oxidative stress occurs due to the accumulation of reactive oxygen species (ROS). ROS can damage DNA, lipids, and proteins (Shields et al. 2021). Superoxide dismutase is an enzyme in a common cellular defense pathway, in which superoxide dismutase converts superoxide radicals to hydrogen peroxide. When cellular defense mechanisms are unable to mitigate ROS formation from mitochondrial respiration and stressors (biological, chemical, radiation), increased ROS levels cause oxidative stress.</span></span></p>
<p>Induction of oxidative stress occurs as a result of an imbalance between the production of radical species and the antioxidant defense systems (Juan et al. 2021). ROS can damage DNA, lipids, and proteins (Shields et al. 2021). Superoxide dismutase is an enzyme in a common cellular defense pathway, in which superoxide dismutase converts superoxide radicals to hydrogen peroxide. When cellular defense mechanisms are unable to mitigate ROS formation from mitochondrial respiration and stressors (biological, chemical, radiation), increased ROS levels cause oxidative stress.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">The biological plausibility linking increases in oxidative stress to reactive oxygen species (ROS) is strong. Reactive oxygen species (ROS) are produced by many normal cellular processes (ex. cellular respiration, mitochondrial electron transport, specialized enzyme reactions) and occur in multiple chemical forms (ex. superoxide anion, hydroxyl radical, hydrogen peroxide). Antioxidant enzymes play a major role in reducing reactive oxygen species (ROS) levels in cells (Ray et al. 2012) to prevent cellular damage to lipids, proteins, and DNA (Juan et al. 2021). Oxidative stress occurs when antioxidant enzymes do not prevent ROS levels from increasing in cells, often induced by environmental stressors (biological, chemical, radiation).</span></span></p>
<p>The biological plausibility linking increases in oxidative stress to reactive oxygen species (ROS) is strong. Reactive oxygen species (ROS) are produced by many normal cellular processes (ex. cellular respiration, mitochondrial electron transport, specialized enzyme reactions) and occur in multiple chemical forms (ex. superoxide anion, hydroxyl radical, hydrogen peroxide). Antioxidant enzymes play a major role in reducing reactive oxygen species (ROS) levels in cells (Ray et al. 2012) to prevent cellular damage to lipids, proteins, and DNA (Juan et al. 2021). Oxidative stress occurs when antioxidant enzymes do not prevent ROS levels from increasing in cells, often induced by environmental stressors (biological, chemical, radiation).</p>
<p style="text-align:center"><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Lu et al. 2016; Alomar et al. 2017; Chen et al. 2017; Veneman et al. 2017; Barboza et al. 2018; Choi et al. 2018; Espinosa et al. 2018</span></span></span></p>
<p style="text-align:center"><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Browne et al. 2013; Jeong et al. 2016, 2017; Paul-Pont et al. 2016; Lei et al. 2018; Yu et al. 2018</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">The accumulation of reactive oxygen species (ROS), and resulting oxidative stress, is well-established (see Shields 2021 for overview). In the studies listed in the above table, changes in enzyme activity and changes in gene expression are the most common oxidative stress effects detected due to increases in reactive oxygen species (see additional study details in table below). Increases in gene expression or enzyme activity of superoxide dismutase, catalase, glutathione peroxidase, and other antioxidants are frequently used as indicators of oxidative stress.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Diet exposure of 0.01, 0.1, 0.5 mg/day of 5 and 20 um polystyrene microplastic particles.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Five-week old male mice showed changes in enzyme levels responsible for eliminating ROS. Decreased catalase at 0.1/0.5 mg/day, increased glutathione peroxidase at all doses, increased superoxide dismutase at all doses.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cerebral and epithelial human cell lines showed measured increased percent effect of ROS (as superoxide generated) with corresponding decreases in cell viability.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 20, 200, 2000 ug/L of 5 and 20 um polystyrene microplastics.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult five-month old fish showed changes in enzyme levels responsible for eliminating ROS. Increased catalase at 200/2000 ug/L, increased superoxide dismutase at all doses.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Survey of wild fish with microplastic ingestion versus no microplastic ingestion.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 0.010, 0.016 mg/L of Mercury chloride, 0.26, 0.69 mg/L of 1-5 um polymer microspheres, and mixture.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Juvenile fish showed increased ROS (Brain and muscle lipid peroxidation levels) and corresponding changes in enzyme levels (increases in muscle lactate dehydrogenase, decreases in isocitrate dehydrogenase). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 50, 250 mg/L of 150-180 um, 300-355 um polyethylene microspheres</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In vitro exposure of 100 mg/L of polyvinylchloride and polyethylene microplastics</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fish head-kidney leucocytes showed increased gene expression of nuclear factor (nrf2), associated with oxidative stress, only statistically significant in S. aurata.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Lugworms showed decreased ability to respond to ROS by ferric reducing antioxidant power (FRAP) assay, statistically significant only with phenanthrene.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 10 ug/mL of 0.05, 0.5, 6 um diameter polystyrene microbeads.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rotifers showed increased ROS levels, changes in phosphorylation of MAPK signaling proteins, and corresponding changes in enzyme and protein levels (decreased glutathione, increased superoxide dismutase, increased glutathione reductase, increased glutathione reductase, glutathione S-transferase). Enzyme statistical significance was seen most frequently with 0.05 diameter size class).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 20 ug/mL of 0.05, 0.5, 6 um diameter polystyrene microbeads.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Copepods showed increased ROS for 0.05 um diameter size class only. Corresponding increases in enzymes were also seen only in 0.05 um diameter size class (glutathione reductase, glutathione peroxidase, glutathione S-transferase, superoxide disumutase).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 30 ug/L fluoranthene, 32 ug/L of 2 and 6 um polystyrene microbeads, and mixture for 7 days and depuration for 7 days.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mussels showed increased ROS production in all treatments for 7 days, changes in enzyme and gene levels were observed for catalase, superoxide dismutase, glutathione S-transferase, glutathione reductase, and lipid peroxidation, statistical significance was not always observed.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yu et al. (2018)</span></span></p>
</td>
</tr>
</tbody>
</table>
<p>1 Assumed: study selected stressor(s) known to elevate reactive oxygen species (ROS) levels, endpoints verified increased oxidative stress and disrupted pathway.</p>
<p> </p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>The reactive oxygen species (ROS) increase needed to elicit oxidative stress is highly dependent on many other variables including age, tissue, sex, nutritional status, and co-exposures to other stressors. It is consistently characterised as an 'excess' of ROS in order to create a state of oxidative stress. Consequently, the quantitative relationship is not easily generalized. </p>
<p><!--EndFragment --></p>
<p>Some examples of normal levels have been reported at 1-8 μM (H2O2) in normal human plasma, while only 1 μM ROS present in healthy cells (Lacy et al. 2000). Inflammatory lung diseases can cause H2O2 excesses to the level of a 20-fold increment. It can also cause the level of H2O2 in ischemia and reperfusion to reach 160 μM (Burgoyne et al. 2013).</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>AP-1 and NF-κB are ROS sensing transcription factors and act as redox sensors due to the presence of a single Cys in their DNA-binding domains (Abate et al. 2006). Oxidation of these Cys residues blocks their binding to the respective consensus DNA sequences. Apurinic/apyrimidinic (AP) endonuclease 1 (APE1), functions as a reducing agent for various transcription factors (Evans et al. 2000). This ubiquitous multifunctional protein is induced by ROS (Ramana et al. 1998) and is involved in base excision repair (Demple and Sung 2005). Although reducing condition is favorable for DNA binding, both AP-1 and NF-κB can be activated by oxidative stress via induction of APE1. A Zn-finger DNA-binding protein, early growth response gene-1 (Egr-1), is activated by ROS, and a positive feedback loop between APE1 and Egr-1 regulates their early transcriptional activation after oxidative stress (Pines et al. 2005). Egr-1 also induces SOD1 and thus reduces free radical-induced damage (Minc et al. 1999).</p>
<h4>References</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Alomar, C., Sureda, A., Capo, X., Guijarro, B., Tejada, S. and Deudero, S. 2017. Microplastic ingestion by Mullus surmuletus Linnaeus, 1758 fish and its potential for causing oxidative stress. Environmental Research 159: 135-142.</span></span></p>
<p>Abate, C., Patel, L., Rauscher III, F. J., & Curran, T. (1990). Redox regulation of fos and jun DNA-binding activity in vitro. Science, 249(4973), 1157-1161.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Barboza, LG.A., Vieira, L.R., Branco, V., Figueiredo, N., Carvalho, F., Carvalho, C., and Guilhermino, L. 2018. Microplastics cause neurotoxicity, oxidative damage and energy-related changes and interact with the bioaccumulation of mercury in the European seabass, Dicentrachus labrux (Linneaeus, 1758). Aquatic Toxicology 195: 49-57.</span></span></p>
<p>Alomar, C., Sureda, A., Capo, X., Guijarro, B., Tejada, S. and Deudero, S. 2017. Microplastic ingestion by Mullus surmuletus Linnaeus, 1758 fish and its potential for causing oxidative stress. Environmental Research 159: 135-142.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Browne, M.A. Niven, S.J., Galloway, T.S., Rowland, S.J., and Thompson, R.C. 2013. Microplastic moves pollutants and additives to worms, reducing functions linked to health and biodiversity. Current Biology 23: 2388-2392.</span></span></span></p>
<p>Barboza, LG.A., Vieira, L.R., Branco, V., Figueiredo, N., Carvalho, F., Carvalho, C., and Guilhermino, L. 2018. Microplastics cause neurotoxicity, oxidative damage and energy-related changes and interact with the bioaccumulation of mercury in the European seabass, Dicentrachus labrux (Linneaeus, 1758). Aquatic Toxicology 195: 49-57.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Chen, Q., Gundlach, M., Yang, S., Jiang, J., Velki, M., Yin, D., and Hollert, H. 2017 Quantitative investigation of the mechanisms of microplastics and nanoplastics toward larvae locomotor activity. Science of the Total Environment 584-585: 1022-1031.</span></span></span></p>
<p>Browne, M.A. Niven, S.J., Galloway, T.S., Rowland, S.J., and Thompson, R.C. 2013. Microplastic moves pollutants and additives to worms, reducing functions linked to health and biodiversity. Current Biology 23: 2388-2392.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Choi, J.S., Jung, Y.J., Hong, N.H., Hong, S.H., and Park, J.W. 2018. Toxicological effects of irregularly shaped and spherical microplastics in a marine teleost, the sheepshead minnow (Cyprinodon variegatus). Marine Pollution Bulletin 129: 231-240.</span></span></span></p>
<p>Burgoyne, J. R., Oka, S. I., Ale-Agha, N., & Eaton, P. (2013). Hydrogen peroxide sensing and signaling by protein kinases in the cardiovascular system. Antioxidants & redox signaling, 18(9), 1042-1052.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Deng, Y., Zhang, Y., Lemos, B., and Ren, H. 2017. Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. Science Reports 7: 1-10.</span></span></span></p>
<p>Chen, Q., Gundlach, M., Yang, S., Jiang, J., Velki, M., Yin, D., and Hollert, H. 2017 Quantitative investigation of the mechanisms of microplastics and nanoplastics toward larvae locomotor activity. Science of the Total Environment 584-585: 1022-1031.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Espinosa, C., Garcia Beltran, J.M., Esteban, M.A., and Cuesta, A. 2018. In vitro effects of virgin microplastics on fish head-kidney leucocyte activities. Environmental Pollution 235: 30-38.</span></span></p>
<p>Choi, J.S., Jung, Y.J., Hong, N.H., Hong, S.H., and Park, J.W. 2018. Toxicological effects of irregularly shaped and spherical microplastics in a marine teleost, the sheepshead minnow (Cyprinodon variegatus). Marine Pollution Bulletin 129: 231-240.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Imhof, H.K., Rusek, J., Thiel, M., Wolinska, J., and Laforsch, C. 2017. Do microplastic particles affect Daphnia magna at the morphological life history and molecular level? Public Library of Science One 12: 1-20.</span></span></p>
<p>Demple, B., & Sung, J. S. (2005). Molecular and biological roles of Ape1 protein in mammalian base excision repair. DNA repair, 4(12), 1442-1449.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Jeong, J. and Choi, J. 2020. Development of AOP relevant to microplastics based on toxicity mechanisms of chemical additives using ToxCast™ and deep learning models combined approach. Environment International 137:105557.</span></span></p>
<p>Deng, Y., Zhang, Y., Lemos, B., and Ren, H. 2017. Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. Science Reports 7: 1-10.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Jeong, C.B., Kang, H.M., Lee, M.C., Kim, D.H., Han, J., Hwang, D.S. Souissi, S., Lee, S.J., Shin, K.H., Park, H.G., and Lee, J.S. 2017. Adverse effects of microplastics and oxidative stress-induced MAPK/NRF2 pathway-mediated defense mechanisms in the marine copepod Paracyclopina nana. Science Reports 7: 1-11.</span></span></p>
<p>Espinosa, C., Garcia Beltran, J.M., Esteban, M.A., and Cuesta, A. 2018. In vitro effects of virgin microplastics on fish head-kidney leucocyte activities. Environmental Pollution 235: 30-38.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Jeong, C.B., Wong, E.J., Kang, H.M., Lee, M.C., Hwang, D.S., Hwang, U.K., Zhou, B., Souissi, S., Lee, S.J., and Lee, J.S. 2016. Microplastic size-dependent toxicity, oxidative stress induction, and p-JNK and p-p38 activation in the Monogonout rotifer (Brachionus koreanus). Environmental Science and Technology 50: 8849-8857.</span></span></p>
<p>Evans, A. R., Limp-Foster, M., & Kelley, M. R. (2000). Going APE over ref-1. Mutation Research/DNA Repair, 461(2), 83-108.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Juan, C.A., de la Lastra, J.M.P., Plou, F.J., and Lebena, E.P. 2021. The chemistry of reactive oxygen species (ROS) revisited: Outlining their role in biological macromolecules (DNA, lipids and proteins) and induced pathologies. International Journal of Molecular Sciences 22: 4642.</span></span></p>
<p>Imhof, H.K., Rusek, J., Thiel, M., Wolinska, J., and Laforsch, C. 2017. Do microplastic particles affect Daphnia magna at the morphological life history and molecular level? Public Library of Science One 12: 1-20.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Lei, L., Wu, S., Lu, S., Liu, M., Song, Y., Fu, Z., Shi, H., Raley-Susman, K.M., and He, D. 2018. Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematode Caenorhabditis elegans. Science of the Total Environment 619-620: 1-8.</span></span></p>
<p>Jeong, J. and Choi, J. 2020. Development of AOP relevant to microplastics based on toxicity mechanisms of chemical additives using ToxCast™ and deep learning models combined approach. Environment International 137:105557.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Paul-Pont, I., Lacroix, C., Gonzalez Fernandez, D., Hegaret, H., Lambert, C., Le Goic, N., Frere, L., Cassone, A.L., Sussarellu, R. Fabioux, C., Guyomarch, J., Albentosa, M., Huvet, A., and Soudant, P. 2016. Exposure of marine mussels Mytillus spp. to polystyrene microplastics: Toxicity and influence on fluoranthene bioaccumulation. Environmental Pollution 216: 724-737.</span></span></p>
<p>Jeong, C.B., Kang, H.M., Lee, M.C., Kim, D.H., Han, J., Hwang, D.S. Souissi, S., Lee, S.J., Shin, K.H., Park, H.G., and Lee, J.S. 2017. Adverse effects of microplastics and oxidative stress-induced MAPK/NRF2 pathway-mediated defense mechanisms in the marine copepod Paracyclopina nana. Science Reports 7: 1-11.</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">R<span style="font-size:16px">ay, P.D., Huang, B.-W., and Tsuji, Y. 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signalling. Cellular Signalling 24:981-990.</span></span></span></p>
<p>Jeong, C.B., Wong, E.J., Kang, H.M., Lee, M.C., Hwang, D.S., Hwang, U.K., Zhou, B., Souissi, S., Lee, S.J., and Lee, J.S. 2016. Microplastic size-dependent toxicity, oxidative stress induction, and p-JNK and p-p38 activation in the Monogonout rotifer (Brachionus koreanus). Environmental Science and Technology 50: 8849-8857.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Schrinzi, G.F., Perez-Pomeda, I., Sanchis, J., Rossini, C., Farre, M., and Barcelo, D. 2017. Cytotoxic effects of commonly used nanomaterials and microplastics on cerebral and epithelial human cells. Environmental Research 159: 579-587.</span></span></p>
<p>Juan, C.A., de la Lastra, J.M.P., Plou, F.J., and Lebena, E.P. 2021. The chemistry of reactive oxygen species (ROS) revisited: Outlining their role in biological macromolecules (DNA, lipids and proteins) and induced pathologies. International Journal of Molecular Sciences 22: 4642.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Shields, H.J., Traa, A., and Van Raamsdonk, J.M. 2021. Beneficial and Detrimental Effects of Reactive Oxygen Species on Lifespan: A Comprehensive Review of Comparative and Experimental Studies.</span></span></p>
<p><span style="background-color:#ffffff; color:#222222; font-family:Arial,sans-serif; font-size:13px">Lacy, F., Kailasam, M. T., O’Connor, D. T., Schmid-Schönbein, G. W., & Parmer, R. J. (2000). Plasma hydrogen peroxide production in human essential hypertension: role of heredity, gender, and ethnicity. </span><em>Hypertension</em><span style="background-color:#ffffff; color:#222222; font-family:Arial,sans-serif; font-size:13px">, </span><em>36</em><span style="background-color:#ffffff; color:#222222; font-family:Arial,sans-serif; font-size:13px">(5), 878-884.</span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Veneman, W.J., Spaink, H.P., Brun, N.R., Bosker, T., and Vijver, M.G. 2017. Pathway analysis of systemic transcriptome responses to injected polystyrene particles in zebrafish larvae. Aquatic Toxicology 190: 112-120.</span></span></span></p>
<p>Lei, L., Wu, S., Lu, S., Liu, M., Song, Y., Fu, Z., Shi, H., Raley-Susman, K.M., and He, D. 2018. Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematode Caenorhabditis elegans. Science of the Total Environment 619-620: 1-8.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Yu, P., Liu, Z., Wu, D., Chen, M., Lv, W., and Zhao, Y. 2018. Accumulation of polystyrene microplastics in juvenile Eriocheir sinensis and oxidative stress effects in the liver. Aquatic Toxicology 200: 28-36.</span></span></p>
<p>Marshall, H. E., Merchant, K., & Stamler, J. S. (2000). Nitrosation and oxidation in the regulation of gene expression. The FASEB Journal, 14(13), 1889-1900.</p>
<p>Minc, E., De Coppet, P., Masson, P., Thiery, L., Dutertre, S., Amor-Guéret, M., & Jaulin, C. (1999). The human copper-zinc superoxide dismutase gene (SOD1) proximal promoter is regulated by Sp1, Egr-1, and WT1 via non-canonical binding sites. Journal of Biological Chemistry, 274(1), 503-509.</p>
<p>Paul-Pont, I., Lacroix, C., Gonzalez Fernandez, D., Hegaret, H., Lambert, C., Le Goic, N., Frere, L., Cassone, A.L., Sussarellu, R. Fabioux, C., Guyomarch, J., Albentosa, M., Huvet, A., and Soudant, P. 2016. Exposure of marine mussels Mytillus spp. to polystyrene microplastics: Toxicity and influence on fluoranthene bioaccumulation. Environmental Pollution 216: 724-737.</p>
<p>Pines, A., Bivi, N., Romanello, M., Damante, G., Kelley, M. R., Adamson, E. D., ... & Tell, G. (2005). Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: evidence for an autoregulatory loop. Free radical research, 39(3), 269-281.</p>
<p>Ramana, C. V., Boldogh, I., Izumi, T., & Mitra, S. (1998). Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals. Proceedings of the National Academy of Sciences, 95(9), 5061-5066.</p>
<p>Ray, P.D., Huang, B.-W., and Tsuji, Y. 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signalling. Cellular Signalling 24:981-990.</p>
<p>Schrinzi, G.F., Perez-Pomeda, I., Sanchis, J., Rossini, C., Farre, M., and Barcelo, D. 2017. Cytotoxic effects of commonly used nanomaterials and microplastics on cerebral and epithelial human cells. Environmental Research 159: 579-587.</p>
<p>Shields, H.J., Traa, A., and Van Raamsdonk, J.M. 2021. Beneficial and Detrimental Effects of Reactive Oxygen Species on Lifespan: A Comprehensive Review of Comparative and Experimental Studies.</p>
<p>Veneman, W.J., Spaink, H.P., Brun, N.R., Bosker, T., and Vijver, M.G. 2017. Pathway analysis of systemic transcriptome responses to injected polystyrene particles in zebrafish larvae. Aquatic Toxicology 190: 112-120.</p>
<p>Yu, P., Liu, Z., Wu, D., Chen, M., Lv, W., and Zhao, Y. 2018. Accumulation of polystyrene microplastics in juvenile Eriocheir sinensis and oxidative stress effects in the liver. Aquatic Toxicology 200: 28-36.</p>
<p> </p>
<p> </p>
</div>
<div>
<h4><a href="/relationships/2975">Relationship: 2975: Oxidative Stress leads to Increase, Inflammation</a></h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Life Stage: The life stage applicable to this key event relationship is all life stages. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sex: This key event relationship applies to both males and females.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Taxonomic: This key event relationship appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats) and teleost fish.</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:16px"><span style="font-family:"Calibri",sans-serif">Inflammation is one consequence of oxidative stress. Inflammation can be characterized as a multi-step process (Villeneuve et al. 2018): 1. Activation of tissue cells due to stress; 2. Increases in proinflammatory mediator (ex. cytokines); 3. Leukocyte recruitment; 4. Inflammatory response.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">The biological plausibility linking inflammation to oxidative stress is strong. Oxidative stress triggers cellular signals, mediated by proinflammatory mediators such as cytokines, which initiates inflammation pathways. At the cellular level, there are increases in leukocyte recruitment; at the tissue and organ levels, visible inflammation occurs.</span></span></p>
<p> </p>
<p> </p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Biological, physical, and chemical stressors from environmental sources can increase oxidative stress. Inflammation is one of the most common responses to oxidative stress (for review see Wright and Kelly (2017); Villeneuve et al. (2018); for empirical studies see Gamo et al. (2008); Lu et al. (2016); Jin et al. (2018); Lei et al. (2018)). Stress triggers increased gene response of proinflammatory signaling mediators (ex. cytokines, interleukins, interferons). Increased leukocyte response results in inflammation.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Seven-week old male mice with surgical brain nerve injury showed changes in inflammatory gene expression (increased interleukin-1beta and interleukin 6), with G-protein coupled receptors mitigating the oxidative stress responses.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 20, 200, 2000 μg/L of 70 nm and 5 um polystyrene microplastics.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult 5-month old fish had increased oxidative stress enzyme levels of superoxide dismutase and catalase and liver inflammation.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 100, 1000 ug/L of 0.5 and 50 um diameter polystyrene microplastic.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult 6-month old male fish increased oxidative stress as measured by statistically significant changes to gut microbiota and changes to inflammatory gene expression, with statistically significant increases of interleukin-1alpha, interleukin-1beta, interferon, interleukin-6.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult fish showed increased oxidative stress in intestinal damage, and increased intestinal inflammation for all but polystyrene (PS) exposure.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Lei et al. (2018)</span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<h4>References</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Gamo, K., Kiryu-Seo, S., Konishi, H., Aoki, S., Matushima, K., Wada, K., and Kiyama, H. 2008. G-protein-coupled receptor screen reveals a role for chemokine recepteor CCR5 in suppressing microglial neurotoxicity. Journal of Neuroscience 28: 11980-11988.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Jin, Y., Xia, J., Pan, Z., Yang, J., Wang, W., and Fu, Z. 2018. Polystyrene microplastics induce microbiota dysbiosis and inflammation in the gut of adult zebrafish. Environmental Pollution 235: 322-329.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Lei, L., Wu, S., Lu, S., Liu, M., Song, Y., Fu, Z., Shi, H., Raley-Susman, K.M., and He, D. 2018. Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematode Caenorhabditis elegans. Science of the Total Environment 619-620: 1-8.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Lu, Y., Zhang, Y., Dengy, Y., Jiang, W., Zhao, Y., Geng, J., Ding, L., Ren, H. 2016. Uptake and accumulation of polystyrene microplastics in zebrafish (Danio rerio) and toxic effects in liver. Environmental Science and Technology 50: 4054-4060.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Villeneuve, D.L., Landesmann, B., Allavena, P., Ashley, N., Bal-Price, A., Corsini, E., Halappanavar, S., Hussell, T., Laskin, D., Lawrence, T., Nikolic-Paterson, D., Pallary, M., Paini, A., Pietrs, R., Roth, R., and Tschudi-Monnet, F. 2018. Toxicological Sciences 346:352.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Wright, S.L. and Kelly, F.J. 2017. Plastic and human health: a micro issue? Enviromental Science and Technology 51: 6634-6647.</span></span></p>
</div>
<div>
<h4><a href="/relationships/2976">Relationship: 2976: Increase, Inflammation leads to General Apoptosis</a></h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Life Stage: The life stage applicable to this key event relationship is all life stages. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sex: This key event relationship applies to both males and females.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Taxonomic: This key event relationship appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats) and teleost fish.</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Pathways leading to apoptosis, or single cell death, have traditionally been studied as both independent and simultaneous from pathways leading to necrosis, or tissue-wide cell death, with both overlap and distinct mechanisms (Elmore 2007). For the purposes of this key event relationship, we are characterizing widespread cell-death due to inflammation (Bock and Riley 2022), while acknowledging that cell death can be caused by multiple stressors, and need not include inflammation.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">The biological plausibility linking apoptosis to inflammation is strong. Inflammation is an indicator for damage, and cell surface markers activate apoptosis pathways for cells that have lost functional capabilities.</span></span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Apoptosis is one of the most common responses to inflammation as a controlled pathway for cell-death due to detected cell damage (for review see Balkwill (2003); Elmore (2007); for empirical studies see Gamo et al. (2008); Lu et al. (2016); Jin et al. (2018)). Generally cell-surface markers indicate damage for T-cell mediated cytotoxic response and phagocytosis; activation of tumor necrosis factor genes enhance cellular response (Elmore 2007). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Seven-week old male mice with surgical brain nerve injury showed changes in inflammatory gene expression (increased interleukin-1beta and interleukin 6) and corresponding increase in apoptosis gene expression (tumor necrosis factor alpha).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 20, 200, 2000 μg/L of 70 nm and 5 um polystyrene microplastics.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult 5-month old fish had increased liver inflammation and liver necrosis.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 100, 1000 ug/L of 0.5 and 50 um diameter polystyrene microplastic.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult 6-month old male fish increased changes to inflammatory gene expression, with statistically significant increases of interleukin-1alpha, interleukin-1beta, interferon, interleukin-6 and corresponding non-significant increase in apoptosis gene expression (tumor necrosis factor alpha).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Jin et al. (2018)</span></span></p>
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<h4>References</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Balkwill, F. 2003. Chemokine biology in cancer. Seminars in Immunology 15: 49-55.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Bock, F.J. and Riley, J.S. 2023. When cell death goes wrong: inflammatory outcomes of failed apoptosis and mitotic cell death. Cell Death and Differentiation 30: 293-303.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Elmore, S. 2007. Apoptosis: A Review of Programmed Cell Death. Toxicologic pathology 35 (4): 495-516.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Gamo, K., Kiryu-Seo, S., Konishi, H., Aoki, S., Matushima, K., Wada, K., and Kiyama, H. 2008. G-protein-coupled receptor screen reveals a role for chemokine recepteor CCR5 in suppressing microglial neurotoxicity. Journal of Neuroscience 28: 11980-11988.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Jin, Y., Xia, J., Pan, Z., Yang, J., Wang, W., and Fu, Z. 2018. Polystyrene microplastics induce microbiota dysbiosis and inflammation in the gut of adult zebrafish. Environmental Pollution 235: 322-329.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Lu, Y., Zhang, Y., Dengy, Y., Jiang, W., Zhao, Y., Geng, J., Ding, L., Ren, H. 2016. Uptake and accumulation of polystyrene microplastics in zebrafish (Danio rerio) and toxic effects in liver. Environmental Science and Technology 50: 4054-4060.</span></span></p>
<p> </p>
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<h4><a href="/relationships/2977">Relationship: 2977: General Apoptosis leads to Increase, Cancer</a></h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Life Stage: The life stage applicable to this key event relationship is all life stages. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sex: This key event relationship applies to both males and females.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Taxonomic: This key event relationship appears to be present broadly, with representative studies focused in mammals (humans, lab mice, lab rats).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Cancer is a general key event for related diseases each exhibiting uncontrolled proliferation of abnormal cells (for review see Hanahan and Weinberg 2011). A cancer often is initially associated with a specific organ, with malignant tumors developing ability to metastasize, or travel to other areas of the body. Most cancers develop from genetic mutations in normal cells; in this key event relationship we are focusing on disruption of apoptosis and necrosis pathways, leading to cancer. Exposure to chemical stressors, radiation, tobacco smoke, or viruses can increase the likelihood that cancer will develop. Pathways leading to apoptosis, or single cell death, have traditionally been studied as both independent and simultaneous from pathways leading to necrosis, or tissue-wide cell death, with both overlap and distinct mechanisms (Elmore 2007). For the purposes of this key event relationship, we are characterizing cancer due to widespread cell-death. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Cancer cells proliferate due to capabilities summarized by Hanahan and Weinberg (2011): </span></span></p>
<ol>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sustained proliferation signaling – by deregulating normal cell signals, cancer cells can sustain chronic proliferation.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Evading growth suppressors – by evading activities of tumor suppressor genes, cancer cells continue to proliferate.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Activating invasion and metastasis – by altering shape and attachment to cells in the extracellular matrix, cancer cells gain ability to move to other locations.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Enabling replicative immortality – by disabling senescence pathways, cancer cells have extended lifespans.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Inducing angiogenesis – by enabling neovasculature, cancer cells receive nutrients and oxygen and get rid of waste products.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Resisting cell death – by evading apotosis and necrosis defense pathways, cancer cells avoid elimination.</span></span></li>
</ol>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">The biological plausibility linking cancer to avoidance of apoptosis is strong. Apoptosis is a series of related pathways that eliminate abnormal cells. </span></span><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Cancer cells proliferate due to evasion of cellular defenses (apoptosis pathways) and tissue-level defenses (necrosis pathways). Specific modifications to cancer cells that enable proliferation rather than elimination are listed under the Key Event Relationship Description. For review see:</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">1. Heinlein and Chang (2004): Role of androgen receptor in apoptosis, loss of androgen pathway function resulting in increases in mammalian prostate cancer.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">2. Hanahan and Weinberg (2011): Biological capabilities gained by cancer cell to enable proliferation of tumor cells and evasion of normal regulating mechanisms of apoptosis and necrosis pathways in mammals.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">3. Pavet et al. (2014): Role of tumor necrosis factor-related apoptosis-inducing ligandin to induce apoptosis in mammalian cells and reduce incidence of cancer.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">4. Vihervaara and Sistonen (2014): Role of increased rate of transcription of heat shock factor 1 in mammalian cancer cells enhancing survival and metastasis, as well as evasion of cellular defenses.</span></span></p>
<strong>Empirical Evidence</strong>
<p>References cited by Jeong and Choi (2020) are review articles and gene expression studies. Empirical studies linking apoptosis to cancer were not provided.</p>
<h4>References</h4>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Elmore, S. 2007. Apoptosis: A Review of Programmed Cell Death. Toxicologic pathology 35 (4): 495-516.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Hanahan, D. and Weinberg, R.A. 2011. Hallmarks of cancer: the next generation. Cell 144(5): 646-674.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Heinlein, C.A. and Chang, C. 2004. Androgen receptor in prostate cancer. Endocrine Reviews 25: 276-308.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Pavet, V., Shlyakhtina, Y., He, T., Ceschin, D.G., Kohonen, P., Perala, M., Kallioniemi, O., and Gronemeyer, H. 2014. Plasminogen activator urokinase expression reveals TRAIL responsiveness and support fractional survival of cancer cells. Cell Death and Disease 5: e1043.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Vihervaara, A. and Sistonen, L. 2014. HSF1 at a glance. Journal of Cell Scientce 127: 261-266.</span></span></p>