<p>Janaina da Silva <em>(Department of General Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil)</em></p>
<p>Reggiani Vilela Gonçalves <em>(Department of Animal Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil)</em></p>
<p>Fabiana Cristina Silveira Alves de Melo <em>(Department of Animal Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil)</em></p>
<p>Mariáurea Matias Sarandy <em>(Department of Animal Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil)</em></p>
<p>Sérgio Luis Pinto da Matta <em>(Department of General Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil and Department of Animal Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil)</em></p>
<p><u>Of the content populated in the AOP-Wiki:</u></p>
<p>Travis Karschnik <em>(General Dynamics Information Technology, Duluth, MN, USA.)</em></p>
<td>Under development: Not open for comment. Do not cite</td>
<td></td>
<td></td>
<td></td>
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</tbody>
</table>
</div>
</div>
<div id="abstract">
</div>
<div id="background">
</div>
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
<p>This AOP was developed as part of an Environmental Protection Agency effort to increase the impact of AOPs published in the peer-reviewed literature, but heretofore unrepresented in the AOP-Wiki, by facilitating their entry and update. The originating work for this AOP was <strong>da Silva, J., Goncalves, R. V., de Melo, F. C. S. A., Sarandy, M. M., & da Matta, S. L. P. (2021). Cadmium exposure and testis susceptibility: A systematic review in murine models. <em>Biological Trace Element Research</em>, <em>199</em>(7), 2663-2676.</strong> This publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages.</p>
<p>The originating authors acknowledged that Cd induces testicular damages however the impact of Cd on the testicular architecture and the mechanisms involved in this damaging process were not clear. They went on to acknowledge that it remains poorly understood if there is a relationship between dose, route, and time of exposure and the injury intensity. Therefore, they conducted a systematic review to assess whether Cd exposure (in any dose, route, and time of exposure) caused significant testicular tissue alterations, including any outcome of testicular histomorphology, as well as molecular, biochemical, and hormonal evaluations in order to understand the mechanisms involved in the histomorphological changes, in murine models. The authors felt this was extremely important in order to provide a direction for future research in this field and the development of decision making for therapeutic alternatives on the treatment of testicular injuries.</p>
<p> </p>
</div>
<div id="development_strategy">
<h3>Strategy</h3>
<div>
<p>The authors perfromed a bibliography search using the electronic databases Medline/PubMed (https://www.ncbi.nlm.nih.gov/pubmed) and Scopus (https://www.scopus.com/<br />
home.uri), on September 21, 2018, at 2:13 p.m. For all databases, the search filters were based on three complementary levels: (i) animals, (ii) testis, and (iii) cadmium,<br />
which were combined by Boolean connectors [AND]. An initial selection based on title and abstract was performed where pre-clinical studies in murine models were included that assessed the Cd effect on testicular architecture that did or did not perform molecular, biochemical, and/or hormonal analyses. All timings, frequencies, routes, and dosages of Cd (and compounds)exposure were eligible for inclusion. The authors excluded studies that didn't evaluate the Cd exposure in the testicular histomorphology of murine models. Data extraction was based on (i) characteristics of publication: authors, publication year, and country; (ii) characteristics of the experimental animals: animal model, age, weight, number of animals, number of animals per group, and number of groups; (iii) exposure: compounds, doses, periodicity of administration, route, duration, and existence of a control group; (iv) main histomorphological outcomes and analyses as well as the main molecular, biochemical, and hormonal results related with the histomorphological alterations; and (v) secondary outcomes. The quality of the studies was assessed by the criteria described on the SYRCLE’s Risk of Bias (RoB) tool (Systematic Review Centre for Laboratory Animal Experimentation) designed specifically for animal studies. Thirty-seven (37) records were included in the systematic review.</p>
<p>The scope of this project was limited to representing the AOP(s) as presented in the originating publication. No editorilization The literature used to support this AOP and its constituent pages began with the originating publication and followed to the primary, secondary, and tertiary works cited therein. </p>
<p>KE and KER page creation and re-use was determined using Handbook principles where page re-use was preferred. Once a baseline level of information was populated for the AOP the authors of the originating publication were contacted for collaboration.</p>
<p>Efforts were made not to editorialize or otherwise add any content to the AOP or its constituent pages that weren’t provided in the primary, secondary, or tertiary literature. In some cases, however, descriptive content was added to pages e.g., assays on a KE page, even if they weren’t specifically provided in the literature stemming from the originating publication.</p>
<p>Taxonomic applicability: Murine models were the focus of the originating publication however the broader concepts likely apply to broader taxonomic groups.</p>
<p>Life stage applicability: The originating publication dealt with adult, reproductively mature organisms since the KEs were investigated in testis tissues and cells.</p>
<p>Sex applicability: Limited to male sex as constrained by testis.</p>
<p>In vitro data is used to support these domains.</p>
<p>Taxonomic applicability: AOP521 is focused on murine models but element imbalance and micromineral mimicry are not limited to this taxon.</p>
<p>Life stage applicability: AOP521 is focused on the adult life stage but element imbalance and micromineral mimicry are not limited to this life stage.</p>
<p>Sex applicability: AOP521 is focused on the Male sex but element imbalance and micromineral mimicry are not limited to this sex.</p>
<p>In vitro data is used to support these domains.</p>
<h4>Key Event Description</h4>
<p>Essential microminerals involved in the formation of structural and intioxidant enzymes are susceptible to disruption, inhibiting body homeostasis (da Silva et al., 2021; Soetan et al., 2010; Gupta and Gupta 2014). A relationship between between certain essential elements and normal testicular development and spermatogensis has been indicated. (Kowal et al., 2010; Liu et al., 2016; do Carmo Cupertino 2017). Further, essential element imbalance can be associated with gonadal dysfunction, microstructural and functional testis damag,e and reproductive disorders (Soetan et al., 2010, Bindari et al., 2013, do Carmo Cupertino 2017).</p>
<p> </p>
<h4>How it is Measured or Detected</h4>
<p>Methods include <strong>X-ray emission</strong>, <strong>secondary ion emission</strong> and <strong>electron energy loss analysis</strong>. Although X-ray microanalysis is the most used method, many biological problems cannot be solved because of its relatively low sensitivity and inability to analyze light elements. New possibilities are offered by Secondary Ion Mass Analysis and Electron Energy Loss Analysis. Analysis by secondary ion emission permits the study of elements at low and even trace element concentration, and even the lightest elements such as hydrogen and beryllium are detected. Electron Energy Loss Analysis makes possible the study of very small volumes, less than 500 A in diameter (Galle et al., 1979).</p>
<h4>References</h4>
<div>Bindari, Y. R., Shrestha, S., Shrestha, N., & Gaire, T. N. (2013). Effects of nutrition on reproduction-A review. <em>Advances in Applied Science Research</em>, <em>4</em>(1), 421-429.</div>
<div> </div>
<div>da Silva, J., Goncalves, R. V., de Melo, F. C. S. A., Sarandy, M. M., & da Matta, S. L. P. (2021). Cadmium exposure and testis susceptibility: A systematic review in murine models. <em>Biological Trace Element Research</em>, <em>199</em>(7), 2663-2676.</div>
<div> </div>
<div>do Carmo Cupertino, M., Novaes, R. D., Santos, E. C., Bastos, D. S. S., Dos Santos, D. C. M., Fialho, M. D. C. Q., & da Matta, S. L. P. (2017). Cadmium-induced testicular damage is associated with mineral imbalance, increased antioxidant enzymes activity and protein oxidation in rats. <em>Life sciences</em>, <em>175</em>, 23-30.</div>
<div> </div>
<div>Galle, P., Berry, J. P., & Lefevre, R. (1979). Microanalysis in biology and medicine. A review of results obtained with three microanalytical methods. <em>Scanning Electron Microscopy</em>, (2), 703-710.</div>
<div>
<div> </div>
<div>
<div>Gupta, U. C., & Gupta, S. C. (2014). Sources and deficiency diseases of mineral nutrients in human health and nutrition: a review. <em>Pedosphere</em>, <em>24</em>(1), 13-38.</div>
<div> </div>
<div>
<div>Kowal, M., Lenartowicz, M., Pecio, A., Gołas, A., Błaszkiewicz, T., & Styrna, J. (2010). Copper metabolism disorders affect testes structure and gamete quality in male mice. <em>Systems biology in reproductive medicine</em>, <em>56</em>(6), 431-444.</div>
<div> </div>
<div>
<div>Liu, J. Y., Yang, X., Sun, X. D., Zhuang, C. C., Xu, F. B., & Li, Y. F. (2016). Suppressive effects of copper sulfate accumulation on the spermatogenesis of rats. <em>Biological trace element research</em>, <em>174</em>, 356-361.</div>
<div> </div>
<div>Soetan, K. O., Olaiya, C. O., & Oyewole, O. E. (2010). The importance of mineral elements for humans, domestic animals and plants: A review. <em>African journal of food science</em>, <em>4</em>(5), 200-222.</div>
<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>
</tr>
<tr>
<td><a href="/aops/384">Aop:384 - Hyperactivation of ACE/Ang-II/AT1R axis leading to chronic kidney disease </a></td>
<td>KeyEvent</td>
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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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<tr>
<td><a href="/aops/409">Aop:409 - Frustrated phagocytosis leads to malignant mesothelioma</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/413">Aop:413 - Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/416">Aop:416 - Aryl hydrocarbon receptor activation leading to lung cancer through IL-6 toxicity pathway</a></td>
<td>KeyEvent</td>
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<tr>
<td><a href="/aops/418">Aop:418 - Aryl hydrocarbon receptor activation leading to impaired lung function through AHR-ARNT toxicity pathway</a></td>
<td>KeyEvent</td>
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<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>
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<tr>
<td><a href="/aops/319">Aop:319 - Binding to ACE2 leading to lung fibrosis</a></td>
<td>KeyEvent</td>
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<tr>
<td><a href="/aops/451">Aop:451 - Interaction with lung resident cell membrane components leads to lung cancer</a></td>
<td>KeyEvent</td>
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<tr>
<td><a href="/aops/476">Aop:476 - Adverse Outcome Pathways diagram related to PBDEs associated male reproductive toxicity</a></td>
<td>MolecularInitiatingEvent</td>
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<td><a href="/aops/492">Aop:492 - Glutathione conjugation leading to reproductive dysfunction via oxidative stress</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/497">Aop:497 - ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
<td>KeyEvent</td>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<tr>
<td><a href="/aops/327">Aop:327 - Excessive reactive oxygen species production leading to mortality (1)</a></td>
<td>MolecularInitiatingEvent</td>
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<tr>
<td><a href="/aops/328">Aop:328 - Excessive reactive oxygen species production leading to mortality (2)</a></td>
<td>MolecularInitiatingEvent</td>
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<td><a href="/aops/329">Aop:329 - Excessive reactive oxygen species production leading to mortality (3)</a></td>
<td>MolecularInitiatingEvent</td>
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<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>
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<tr>
<td><a href="/aops/534">Aop:534 - Succinate dehydrogenase (SDH) inhibition leads to cancer through oxidative stress</a></td>
<td>KeyEvent</td>
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<tr>
<td><a href="/aops/273">Aop:273 - Mitochondrial complex inhibition leading to liver injury</a></td>
<td>KeyEvent</td>
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<tr>
<td><a href="/aops/488">Aop:488 - Increased reactive oxygen species production leading to decreased cognitive function</a></td>
<td>MolecularInitiatingEvent</td>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<tr>
<td><a href="/aops/282">Aop:282 - Adverse outcome pathway on photochemical toxicity initiated by light exposure</a></td>
<td>MolecularInitiatingEvent</td>
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<tr>
<td><a href="/aops/569">Aop:569 - Decreased DNA methylation of FAM50B/PTCHD3 leading to IQ loss of children via PI3K-Akt pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/613">Aop:613 - Peroxisome proliferator-activated receptor alpha activation leading to early life stage mortality via increased reactive oxygen species production</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/622">Aop:622 - Calcineurin inhibitor induced nephrotoxicity leading to kidney failure</a></td>
<p>ROS is a normal constituent found in all organisms.</p>
<p>ROS is a normal constituent found in all organisms, <em>lifestages, and sexes.</em></p>
<h4>Key Event Description</h4>
<p>Biological State: increased reactive oxygen species (ROS)</p>
<p>Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.</p>
<p>Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
<p>Reactive oxygen species (ROS) are O<sub>2</sub>- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). </p>
<div>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Reactive oxygen species (ROS) refers to the chemical species superoxide, hydrogen peroxide, and their secondary reactive products. In the biological context, ROS are signaling molecules with important roles in cell energy metabolism, cell proliferation, and fate. Therefore, balancing ROS levels at the cellular and tissue level is an important part of many biological processes. Disbalance, mainly an increase in ROS levels, can cause cell dysfunction and irreversible cell damage.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are produced from both exogenous stressors and normal endogenous cellular processes, such as the mitochondrial electron transport chain (ETC). Inhibition of the ETC can result in the accumulation of ROS. Exposure to chemicals, heavy metal ions, or ionizing radiation can also result in increased production of ROS. Chemicals and heavy metal ions can deplete cellular antioxidants reducing the cell’s ability to control cellular ROS and resulting in the accumulation of ROS. Cellular antioxidants include glutathione (GSH), protein sulfhydryl groups, superoxide dismutase (SOD). </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are radicals, ions, or molecules that have a single unpaired electron in their outermost shell of electrons, which can be categorized into two groups: free oxygen radicals and non-radical ROS [Liou et al., 2010]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">highly reactive lipid- or carbohydrate-derived carbonyl compounds</span></span></p>
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</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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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 - Neuron defect induced early behavioral change</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>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/616">Aop:616 - organic UV filter and its Photoproducts reproductive toxicity pathways </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/622">Aop:622 - Calcineurin inhibitor induced nephrotoxicity leading to kidney failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/625">Aop:625 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/628">Aop:628 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated oxidative stress</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>
<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> </p>
</td>
<td>
<p>0, 50,100 and 200 µM of Uranyl Acetate </p>
<p> </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>“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>“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 ofmitochondrial 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>0, 50, 100, or 200 <em>μ</em>M Uranyl Acetate</p>
<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>Strong/easy</p>
medium</td>
<p> </p>
</td>
<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>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>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>
<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>
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<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">Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH oxidase, and redox signaling in the vasculature”, <em>Antioxidants & redox signaling,</em> Vol. 19/10<strong>,</strong> </span><span style="background-color:white"><span style="color:black">Mary Ann Liebert, Larchmont, </span></span><a href="https://doi.org/10.1089/ars.2012.4641" style="color:#0563c1; text-decoration:underline"><span style="background-color:white">https://doi.org/10.1089/ars.2012.4641</span></a></span></span></p>
<p>Lu, C., G. Song, and J. Lin (2006), “Reactive oxygen species and their chemiluminescence-detection methods”, TrAC Trends in Analytical Chemistry, Vol. 25/10, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.trac.2006.07.007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.trac.2006.07.007</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">Ping, Z. et al. (2020), “Oxidative Stress in Radiation-Induced Cardiotoxicity”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2020, Hindawi, </span></span></span><a href="https://doi.org/10.1155/2020/3579143" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1155/2020/3579143</span></span></a></span></span></p>
<p>Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH oxidase, and redox signaling in the vasculature”, Antioxidants & redox signaling, Vol. 19/10, Mary Ann Liebert, Larchmont, <a href="https://doi.org/10.1089/ars.2012.4641" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2012.4641</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">Powers, S.K. and M.J. Jackson. (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, <em>Physiological Reviews,</em> Vol. 88/4<strong>,</strong> American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/physrev.00031.2007" 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/physrev.00031.2007</span></span></a></span></span></p>
<p>Ping, Z. et al. (2020), “Oxidative Stress in Radiation-Induced Cardiotoxicity”, Oxidative Medicine and Cellular Longevity, Vol. 2020, Hindawi, <a href="https://doi.org/10.1155/2020/3579143" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2020/3579143</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">Raimondi, V., F. Ciccarese and V. Ciminale. (2020), “Oncogenic pathways and the electron transport chain: a dangeROS liason”, <em>British Journal of Cancer, </em>Vol. 122/2, Nature Portfolio, London, </span></span></span><a href="https://doi.org/10.1038/s41416-019-0651-y" 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/s41416-019-0651-y</span></span></a></span></span></p>
<p>Powers, S.K. and M.J. Jackson. (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, Physiological Reviews, Vol. 88/4, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00031.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/physrev.00031.2007</a> </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">Seen, S. and L. Tong. (2018), “Dry eye disease and oxidative stress”, <em>Acta Ophthalmologica,</em> Vol. 96/4<strong>,</strong> John Wiley & Sons, Inc., Hoboken, </span><a href="https://doi.org/10.1111/aos.13526" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1111/aos.13526</a></span></span></p>
<p>Raimondi, V., F. Ciccarese and V. Ciminale. (2020), “Oncogenic pathways and the electron transport chain: a dangeROS liason”, British Journal of Cancer, Vol. 122/2, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41416-019-0651-y" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41416-019-0651-y</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">Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, <em>The Journals of Gerontology Series A: Biological Sciences and Medical Sciences</em>, Vol. 68/12, Oxford University Press, Oxford, </span></span></span><a href="https://doi.org/10.1093/gerona/glt057." style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1093/gerona/glt057.</span></span></a> </span></span></p>
<p>Seen, S. and L. Tong. (2018), “Dry eye disease and oxidative stress”, Acta Ophthalmologica, Vol. 96/4, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1111/aos.13526" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/aos.13526</a> </p>
<p style="margin-left:48px"> </p>
<p>Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, Vol. 68/12, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/gerona/glt057." rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/gerona/glt057.</a> </p>
<p> </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">Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, <em>Life, </em>Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, </span></span></span><a href="https://doi.org/10.3390/life11111269" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.3390/life11111269</span></span></a></span></span></p>
<p>Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, Life, Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/life11111269" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/life11111269</a> </p>
<p style="margin-left:48px"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Wang, H. et al. (2019), “Radiation-induced heart disease: a review of classification, mechanism and prevention”, <em>International Journal of Biological Sciences, </em>Vol. 15/10, Ivyspring International Publisher, Sydney, <a href="https://doi.org/10.7150/ijbs.35460" style="color:#0563c1; text-decoration:underline">https://doi.org/10.7150/ijbs.35460</a> </span></span></p>
<p>Wang, H. et al. (2019), “Radiation-induced heart disease: a review of classification, mechanism and prevention”, International Journal of Biological Sciences, Vol. 15/10, Ivyspring International Publisher, Sydney, <a href="https://doi.org/10.7150/ijbs.35460" rel="noreferrer noopener" target="_blank">https://doi.org/10.7150/ijbs.35460</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:#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>
<td><a href="/aops/329">Aop:329 - Excessive reactive oxygen species production leading to mortality (3)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/413">Aop:413 - Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/492">Aop:492 - Glutathione conjugation leading to reproductive dysfunction via oxidative stress</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/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/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/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/615">Aop:615 - Suppression of Keap1 cysteine oxidation leading to liver inflammation</a></td>
<p dir="ltr">ROS is a normal constituent found in all organisms, therefore, all organisms containing lipid membranes may be affected by lipid peroxidation. </p>
<p>Structure: Regardless of sex or life stage, when exposed to free radicals, there is potential for lipid peroxidation as a auxiliary response where there are lipid membranes.</p>
<h4>Key Event Description</h4>
<p>Lipid peroxidation is the direct damage to lipids in the membrane of the cell or the membranes of the organelles inside the cells. Ultimately the membranes will break due to the build-up damage in the lipids. This is mainly caused by oxidants which attack lipids specifically, since these contain carbon-carbon double bonds. During lipid peroxidation several lipid radicals are formed in a chain reaction. These reactions can interfere and stimulate each other. Antioxidants, such as vitamin E, can react with lipid peroxy radicals to prevent further damage in the cell (Cooley et al. 2000).</p>
<h4>How it is Measured or Detected</h4>
<p>The main product of lipid peroxidation, malondialdehyde and 4-hydroxyalkenals, is used to measure the degree of this process. This is measured by photocolorimetric assays, quantification of fatty acids by gaseous liquid chromatography (GLC) or high performance (HPLC) (L. Li et al. 2019; Jin et al. 2010a) or through commercial kits purchased from specialized companies.</p>
<p> </p>
<h4>References</h4>
<p>Cooley HM, Evans RE, Klaverkamp JF. 2000. Toxicology of dietary uranium in lake whitefish (Coregonus clupeaformis). Aquatic Toxicology. 48(4):495–515. https://doi.org/10.1016/S0166-445X(99)00057-0</p>
<p>Jin, Yuanxiang, Xiangxiang Zhang, Linjun Shu, Lifang Chen, Liwei Sun, Haifeng Qian, Weiping Liu, and Zhengwei Fu. 2010a. “Oxidative Stress Response and Gene Expression with Atrazine Exposure in Adult Female Zebrafish (Danio Rerio).” Chemosphere 78 (7): 846–52.</p>
<p>Li, Luxiao, Shanshan Zhong, Xia Shen, Qiujing Li, Wenxin Xu, Yongzhen Tao, and Huiyong Yin. 2019. “Recent Development on Liquid Chromatography-Mass Spectrometry Analysis of Oxidized Lipids.” Free Radical Biology & Medicine 144 (November): 16–34.</p>
<h4><a href="/events/2206">Event: 2206: Increased, histomorphological alteration of testis</a></h4>
<h5>Short Name: Increased, histomorphological alteration of testis</h5>
<p>Taxonomic applicability: AOP521 is focused on murine models but element imbalance and micromineral mimicry are not limited to this taxon.</p>
<p>Life stage applicability: Limited to adult organisms with reproductive tissues and structures.</p>
<p>Sex applicability: Limited to male organisms.</p>
<p>In vitro data is used to support these domains.</p>
<h4>Key Event Description</h4>
<p>Testicular histomorphological alterations include any and all changes to the morphology of cells and tissues associated with the testis. For example: hemorrhage, edema, fibrosis, necrosis, inflammation, calcification, percent interstitial tissue, lymphatic space volume, nuclei volume, thrombosis, vacuolization, tubular diameter changes, and vasculitis. Histomorphological alterations precede decreases in spermatogenesis and steroidogenesis resulting in reproductive failure (da Silva 2021). </p>
<h4>How it is Measured or Detected</h4>
<p>Can be measured quantitatively or qualitatively in any of the testis constituent tissues and cells including interstitial tissue, seminiferous tubules, lumen, albuginea, tunica propria, Leydig cells, blood vessels, and the germinal epithelium. Techniques mostly use staining or microscopy techniques and include the following examples:</p>
<ul>
<li>Annexin-V PI staining</li>
<li>Spectrophotometric hemoglobin assay</li>
<li>TGF-β-dependent gene expression</li>
</ul>
<h4>References</h4>
<div>da Silva, J., Goncalves, R. V., de Melo, F. C. S. A., Sarandy, M. M., & da Matta, S. L. P. (2021). Cadmium exposure and testis susceptibility: A systematic review in murine models. <em>Biological Trace Element Research</em>, <em>199</em>(7), 2663-2676.</div>
<p>Taxonomic Applicability: The relevance for invertebrates has not been evaluated. </p>
<p>Life Stage Applicability: Only applicable for sexually mature adults</p>
<p>Sex Applicability: Only applicable to males</p>
<p><em>In vitro data is used to support these domains.</em></p>
<h4>Key Event Description</h4>
<p dir="ltr"><strong>Spermatogenesis is a multiphase process of cellular transformation that produces mature male gametes known as sperm for sexual reproduction (Xu et al., 2015). The process of spermatogenesis can be broken down into 3 phases: the mitotic proliferation of spermatogonia, meiosis, and post-meiotic differentiation(spermiogenesis) (Boulanger et al., 2015). Spermatogenesis can be impaired within these phases or due to external factors such as chemical exposures or the gonadal tissue environment. For example, zebrafish and fathead minnow exposed to flutamide, an antiandrogen, have shown signs of impaired spermatogenesis such as spermatocyte degradation(Jensen et al., 2004, Yin et al., 2017).</strong></p>
<h4>How it is Measured or Detected</h4>
<p dir="ltr"><strong>Impairment of spermatogenesis can be measured and detected in a multitude of ways. One example of this is qualitative histological assessments (Jensen et al., 2004). Through histology, sperm morphology can be examined and quantified through the number and stage of the sperm. Sperm morphology, overall quantity, and quantity within each stage can be ways to detect impaired spermatogenesis(Uhrin et al., 2000, Xie et al., 2020). Additionally, sperm quality can also be another assessment of impaired spermatogenesis such as sperm motility, velocity, ATP content, and lipid peroxidation(Gage et al., 2004, Xia et al., 2018, Chen et al., 2015). Impaired spermatogenesis can also be seen by measuring sperm density(Chen et al., 2015).</strong></p>
<h4>References</h4>
<p dir="ltr"><strong>Boulanger, G., Cibois, M., Viet, J., Fostier, A., Deschamps, S., Pastezeur, S., Massart, C., Gschloessl, B., Gautier-Courteille, C., & Paillard, L. (2015). Hypogonadism Associated with Cyp19a1 (Aromatase) Posttranscriptional Upregulation in Celf1 Knockout Mice. Molecular and cellular biology, 35(18), 3244–3253. <a href="https://doi.org/10.1128/MCB.00074-15">https://doi.org/10.1128/MCB.00074-15</a></strong></p>
<p dir="ltr"><strong>Chen, J., Xiao, Y., Gai, Z., Li, R., Zhu, Z., Bai, C., Tanguay, R. L., Xu, X., Huang, C., & Dong, Q. (2015). Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects. Aquatic toxicology (Amsterdam, Netherlands), 169, 204–214. https://doi.org/10.1016/j.aquatox.2015.10.020</strong></p>
<p dir="ltr"><strong>Golshan, M. & S.M.H. Alvai (2019) “Androgen signaling in male fishes: Examples of anti-androgenic chemicals that cause reproductive disorders”, Theriogenology, Vol. 139, Elsevier, pp. 58-71. https://doi.org/10.1016/j.theriogenology.2019.07.020 </strong></p>
<p dir="ltr"><strong>Jensen, K.M. et al. (2004) “Characterization of responses to the antiandrogen flutamide in a short-term reproduction assay with the fathead minnow”, Aquatic Toxicology, Vol. 70(2), Elsevier, pp. 99-110. https://doi.org/10.1016/j.aquatox.2004.06.012 </strong></p>
<p dir="ltr"><strong>Uhrin, P., Dewerchin, M., Hilpert, M., Chrenek, P., Schöfer, C., Zechmeister-Machhart, M., Krönke, G., Vales, A., Carmeliet, P., Binder, B. R., & Geiger, M. (2000). Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility. The Journal of clinical investigation, 106(12), 1531–1539. <a href="https://doi.org/10.1172/JCI10768">https://doi.org/10.1172/JCI10768</a></strong></p>
<p dir="ltr"><strong>Xia, H., Zhong, C., Wu, X., Chen, J., Tao, B., Xia, X., Shi, M., Zhu, Z., Trudeau, V. L., & Hu, W. (2018). Mettl3 Mutation Disrupts Gamete Maturation and Reduces Fertility in Zebrafish. Genetics, 208(2), 729–743. https://doi.org/10.1534/genetics.117.300574</strong></p>
<p dir="ltr"><strong>Xie, H., Kang, Y., Wang, S., Zheng, P., Chen, Z., Roy, S., & Zhao, C. (2020). E2f5 is a versatile transcriptional activator required for spermatogenesis and multiciliated cell differentiation in zebrafish. PLoS genetics, 16(3), e1008655. https://doi.org/10.1371/journal.pgen.1008655</strong></p>
<p dir="ltr"><strong>Xu, K., Wen, M., Duan, W., Ren, L., Hu, F., Xiao, J., Wang, J., Tao, M., Zhang, C., Wang, J., Zhou, Y., Zhang, Y., Liu, Y., & Liu, S. (2015). Comparative analysis of testis transcriptomes from triploid and fertile diploid cyprinid fish. Biology of reproduction, 92(4), 95. <a href="https://doi.org/10.1095/biolreprod.114.125609">https://doi.org/10.1095/biolreprod.114.125609</a></strong></p>
<p><strong>Yin, P. et al. (2017) “Diethylstilbestrol, flutamide and their combination impaired the spermatogenesis of male adult zebrafish through disrupting HPG axis, meiosis and apoptosis”, Aquatic Toxicology, Vol. 185, Elsevier, pp. 129-137. https://doi.org/10.1016/j.aquatox.2017.02.013 </strong></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><em><strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Taxonomic applicability</span></span></strong></em><strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">: </span></span></strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Decrease in viable offspring may have relevance for species with sexual reproduction, including fish, mammals, amphibians, reptiles, birds, and invertebrates.</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><em><strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Life stage applicability</span></span></strong></em><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">: Decrease in viable offspring is relevant for reproductively mature individuals. </span></span></span></span></span></p>
<p><strong><em><span style="font-size:11.0pt"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Sex applicability</span></span></span></em></strong><span style="font-size:11.0pt"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">: Decrease in viable offspring can be measured for both males and females.</span></span></span></p>
<p><em>In vivo data is used to support these domains.</em></p>
<h4>Key Event Description</h4>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">The production of viable offspring in sexual reproduction is through fertilization of oocytes that then develop into offspring. Producing viable offspring is dependent on multiple factors, including but not limited to, oocyte maturation and ovulation, spermatogenesis and sperm production, successful fertilization of oocytes, development including successful organogenesis, and adequate nutrition. </span></span></span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Effects on the production of viable offspring is measured or detected through the ability (or inability) of reproductively mature organisms to produce offspring, number of offspring produced (per pair, individual, or population), and/or percent of fertilized, viable embryos. </span></span></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/3115">Relationship: 3115: Increased, essential element imbalance leads to Increased, Reactive oxygen species</a></h4>
<h4><a href="/relationships/3115">Relationship: 3115: Increased, essential element imbalance leads to Increase, ROS</a></h4>
<p>Essential elements including copper, zinc, and iron are required for normal cellular processes and therefore are subject to mechanisms which regulate their function. In contrast, nonessential elements e.g., mercury, cadmium, and lead don’t have known nutritive value and accordingly, no dedicated mechanisms have evolved for their uptake in most animal species (Bridges and Zalups 2005). In spite of this, these toxic metals still enter various cells (Clarkson 1993, Ballatori 2002, Zalups 2000, Zalups and Ahmad 2003).</p>
<p>The concept of mimicry, in both molecular and ionic forms, has been hypothesized as mechanisms by which these metal species can enter target cells. Molecular mimicry refers to the bonding of metal ions to nucleophilic groups on certain biomolecules results in the formation of organo-metal complexes that can behave or serve as a structural and/or functional homolog of other endogenous biomolecules or the molecule to which the metal ion has bonded (Clarkson 1993, Ballatori 2002, Zalups 2000). Alternatively, ionic mimicry refers to the ability of an unbound, native, cationic species of a metal to mimic an essential element or cationic form of that element (Clarkson 1993, Wetterhahn-Jennette 1981, Zalups and Ahmad 2003). Either type of mimic may also be classified as structural or functional mimics. A structural mimic refers to an elemental or molecular species that is similar in size and shape to another element or molecule. A functional mimic is one that can elicit the same effect, i.e., physiological response, as the native element or molecule (Bridges and Zalups 2005).</p>
<p>Essential element imbalance, resulting from molecular and ionic mimicry, causing either deficiency or overload, can inhibit the antioxidant ability of the elements that are mimicked i.e., Selenium, Zinc, Copper, Magnesium, and Manganese, among others.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The primary antioxidant defense systems are enzymatic reaction systems in the body, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), etc. (Erdogan et al., 2005, Valdivia et al., 2007). Low molecular weight, non-enzymatic antioxidants also play an important role in the antioxidant protective system by assisting enzyme activity. These include glutathione, pantothenic acid, vitamins and minerals, such as zinc, selenium, and copper (Agarwal et al., 2004, Wolksi 2011). Cadmium exposure has been related to an increase of ROS and to induction of oxidative stress through indirect mechanisms: the first consists in Cd binding to sulfhydryl groups of ROS scavengers, which determines an alteration of their regulatory activity (Stohs and Bagchi 1995, Valko et al., 2005), and the second is driven by interference between Cd and selenium, with major affected targets being the glutathione (GSH) system and, particularly, the GSH peroxidase (GSH-Px) (Ren et al., 2012, Sugawara et al., 1986, Omaye and Tappel 1975, Sugawara and Sugawara 1984, Li et al., 2010, Abarikwu et al., 2013, Yiin et al., 1999). Both processes result in the production of ROS, such as superoxide ion, hydrogen peroxide and hydroxyl radicals (Stohs and Bagchi 1995, Valko et al., 2005). Selenium is a structural component of selenoproteins, comprising antioxidant enzymes, such as GSH-Px (Dodig and Cepelak, Flohe et al., 1973) and which catalyzes the reduction of hydrogen peroxide and organic peroxides, including phospholipids peroxides. Cadmium can also replace calcium in calcium-binding proteins, causing disruption or cessation of activity, which can lead to oxidative stress (El-Demerdash et al., 2004).</p>
<p>Cd interaction with ROS scavengers is mainly mediated by the displacement of Zn and Cu from antioxidant enzymes. This is a molecular mimicry which also precipitates conformational changes and impairment in the activity of the enzymes. Additionally, the increased Cu concentration in the cell also induces ROS production (Pillai and Gupta 2005, Yang et al., 2000, Hanna and Mason 1992). Another mechanism of Cd-induced oxidative stress is related to Cd interference with Se, and consequent interference with reduced GSH, oxidized GSH, GSH-Px, GSH reductase and catalase activities. Another route Cd may induce oxidative damage is by enhancing peroxidation of membrane lipids and altering the antioxidant system of the cells (Sarkar et al. 1995)</p>
<p>Cd and Zinc share similar chemical properties and bind to biological macromolecules containing sulphydryl, hydroxyl and nitroxyl groups. Although Cd ion is larger than Zn ion, Cd has a higher affinity for sulphydryl-containing proteins and nucleic acids, and substitutes for Zn through molecular mimicry, in the presence of excess Cd (Jacobson and Turner 1980). Based on their similarities Cd can potentially interfere with several Zn-mediated biological processes. Zinc is an antioxidant essential trace mineral that acts by neutralizing free radical generation (Powell 2000). Zn protection against the cytotoxicity of Cd may be related to the maintenance of normal redox balance inside the cell (Souza et al. 2004). Bray and Bettger 1990 suggested Zn could exert a direct antioxidant action by occupying the iron or copper binding sites of lipids, proteins, and DNA. Further, zinc-deficient male rats, as a result of Cd exposure, had higher levels of LPO, protein oxidation, and decreased SOD activity, which lead to reduced testicular growth and oxidative stress (Oteiza et al., 1999).</p>
<p>Thévenod 2009 indicated that the effect of Cd on the cellular antioxidant enzymatic system is mediated by inhibition of the mitochondrial electron transport chain. Furthermore, displacement of redox active metals has also been proposed to explain oxidative stress and the antioxidant reaction in response to Cd toxicity (Cupertino et al., 2017).</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Rats exposed to Cd displayed significant increases in MDA and metallothionein concentrations in the testes. Cadmium exposure significantly decreased the GPx, mitochondrial Mn-SOD, cytosolic CuZn-SOD, and CAT activities in the testes.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Cd induced a reduction in liver and kidney GPx, CuZn-SOD, Mn-SOD and CAT activities. Moreover, Cd exposure increased hepatic and renal MDA concentration. The same treatment increased the 8-oxodGuo level in liver and kidney. Our investigations reported that cadmium toxicity implicated probably reactive oxygen spices leading to oxidative stress in rat tissues confirmed by the decrease of antioxidant enzymes activity and the LPO. </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">To analyze the role of Nrf2 in the toxic response to Cd, mouse embryonic fibroblast cells (MEF) derived from Nrf2 wild-type (Nrf2+/+) and knockout (Nrf2-/-) mice were treated with Cd, and the production of superoxide anion radical was measured. Cd induced oxidative stress in both wild-type and Nrf2-null cells dose dependently.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Cd treatment doubled the basal ESR signal for POBN adducts. Acute Cd exposure induces in vivo hepatic free radical generation as evidenced by POBN-trapped radical metabolites formed in the liver and excreted into the bile.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">There was a significant increase in lipid peroxidation products in testes following treatment with all doses of metal used beside the 25 μg/kg Cd.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Rats (31-day-old) were injected s.c. with a single dose of either saline or CdCl2. By 48 h post-injection there was a marked increase in TBARS concentration, and a marked decrease in glutamine synthetase activity. Testes TBARS concentrations were significantly higher in the zinc-deficient rats than in controls.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">The results from this analysis show that Cd at the various concentrations (50–750 μmol/L) elevated the MDA level/LPO rate in bull sperm suspensions in a concentration-dependent manner. There was a positive correlation between the concentration of Cd and the LPO rate.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">After 8 h of Cd treatment, steady-state levels of MT-1 mRNA, GST-α mRNA, and γ-GCS mRNA increased approximately 23-, 5-, and 3-fold, respectively, compared to their mRNA levels in cells that were not exposed to Cd. However, the expression of γ-GCS expression returned to control levels after 24 h of treatment whereas MT-1 and GST-α expression did not. The GST-α mRNA level in Cd-exposed cells was elevated to approximately the same extent above control at both time points. In contrast, the magnitude of MT-1 induction in Cd-exposed cells was 90% lower at 24 h than at 8 h.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Cadmium induced a dose-related increase in lung LPO as measured by total lung TBARS.</span></span></span></p>
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</table>
<strong>Uncertainties and Inconsistencies</strong>
<p>Liu et al., 2009 showed that, contrary to direct ESR evidence for ROS generation following acute Cd overload, ESR evidence for free radical generation following long-term, low-dose Cd exposure is often obscure. They showed that mice given a diet containing 100 ppm CdCl2 for 6 months, followed by injection of POBN did not enhance the POBN-trapped radicals in the liver and kidneys, nor did it increase the hepatic and renal lipid peroxidation levels.</p>
<p>Other examples of a lack of ROS production following chronic Cd exposures are characterized as follows. </p>
<ul>
<li>A prolonged Cd exposure (100 ppm, 23 weeks) through the drinking water didn’t produce overt changes in cellular redox status and lipid peroxidation levels (Thijssen et al., 2007).</li>
<li>Dietary Cd exposure (up to 80 ppm) for one year even decreases lipid peroxidation levels in the liver and kidney of the bank vole (Wlostowski et al., 2000).</li>
<li>A single oral dose of Cd (20 mg/kg) initially increased hepatic lipid peroxidation levels and iron concentrations 5 hr after Cd administration in mice, but repeated oral doses (10 mg/kg, daily for 14 days) produced no change or a slightly decrease in hepatic lipid peroxidation levels (Djukic-Dosic et al., 2008).</li>
<li>ROS tolerance is also seen with a long-term (one year) injection of Cd at low levels (0.3 mg/kg, 3 days/week), without increases in tissue lipid peroxidation levels (Kamiyama et al., 1995).</li>
<li>In rats given chronic Cd injections (0.6 mg/kg for 12 weeks), kidney injury is evident with dramatic increase in expression of kidney injury molecule-1 and MT (Prozialeck et al., 2007), but the changes in the expressions of ROS-related genes and oxidative DNA damage genes are not appreciable.</li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">Induction of ROS required 50 μM Cd in the wild type. Markedly elevated ROS production was observed in Nrf2-null cells treated with as low as 2 μM d, indicating that loss of Nrf2 function increased oxidative stress in cells both under basal conditions and in the presence of Cd.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">There was a significant increase in lipid peroxidation products in testes following treatment with all doses of metal used beside the 25 μg/kg Cd.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">Cadmium induced a dose-related increase in lung LPO as measured by total lung TBARS but significant differences were only seen following the administration of 250 and 1000 ug/kg bodyweight.</span></span></span></span></p>
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<strong>Time-scale</strong>
<p>See UNCERTAINTIES AND INCONSISTENCIES section for information about some chronic exposure outcomes.</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">Measurements at a single time, 30 days.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">Measurements at a single time, 4 weeks</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">Measurements at a single time, 6 hours</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">Measurements at a single time, 40-60 minutes (See UNCERTAINTIES AND INCONSISTENCIES section for information about some chronic exposure outcomes)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">A significant rise was noted at 72 h with the 25 μg dose. The 500 μg/kg Cd dose markedly altered testes lipid peroxidation at 24 and 72 h</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">48 hours was the shortest time period eliciting marked increase in TBARS and decrease in glutaimen synthetase activity. Zinc deficient rats showed significant differences in TBARS and glutamine synthetase activity after 24 hours.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">Measurements at a single time, 60 minutes after injection</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">After 8 h of Cd treatment, steady-state levels of MT-1 mRNA, GST-α mRNA, and γ-GCS mRNA increased approximately 23-, 5-, and 3-fold, respectively, compared to their mRNA levels in cells that were not exposed to Cd. However, the expression of γ-GCS expression returned to control levels after 24 h of treatment whereas MT-1 and GST-α expression did not.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="color:black">Measurements at a single time, 24 hours</span></span></span></span></p>
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</table>
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<p>Zalups, R. K., and Ahmad, S. (2003). Molecular handling of cadmium in transporting epithelia. Toxicology and applied pharmacology, 186(3), 163-188.</p>
</div>
<div>
<h4><a href="/relationships/2009">Relationship: 2009: Increased, Reactive oxygen species 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/3116">Relationship: 3116: Oxidative Stress leads to Increased, LPO</a></h4>
<h4><a href="/relationships/3116">Relationship: 3116: Increase, Oxidative Stress leads to Increase, LPO</a></h4>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><strong>Taxonomic Applicability</strong>: Spermatogenesis is one of the most conserved biological processes from <em>Drosophila </em>to humans (Wu et al., 2016). As a result, animals who utilize sexual reproduction as their way to produce offspring are heavily reliant on spermatogenesis being effective and normal. There are studies on reproduction and spermatogenesis across a multitude of taxa. </span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><strong>Sex Applicability</strong>: Spermatogenesis is a male-specific process (<span style="color:#212529">Schulz et al., 2010, </span>Tang et al., 2018, Wu et al., 2015 ). Thus, the present relationship is only relevant for males.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><strong>Life Stage Applicability</strong>: Spermatogenesis and reproduction are only relevant for sexually-mature adults.</span></span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:12.0pt"><span style="font-family:"Arial",sans-serif">Spermatogenesis is a multiphase process of cellular transformation that produces mature male gametes known as sperm for sexual reproduction. The process of spermatogenesis can be broken down into 3 phases: the mitotic proliferation of spermatogonia, meiosis, and post meiotic differentiation (spermiogenesis) (Boulanger et al., 2015). Male fertility is dependent on the quantity as well as the proper cellular morphology of the sperm formed in the testes. The fusion of sperm and oocytes is the key step for the beginning of life known as fertilization. Oocyte fertilization and the production of viable offspring from sexual reproduction are dependent on spermatogenesis and sufficient quantity and quality of sperm. When the impairment of spermatogenesis occurs, it can result in impaired reproduction with a decrease in viable offspring.</span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in sperm density of F1 and F2 males compared to control</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decreased sperm quality as measured by motility, velocity, ATP content and lipid peroxidation in F1 and F2 males</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Delayed hatching at 48hpf and increased malformation and mortality were observed in the offspring from BPA- exposed F2; paternal-specific resulting from BPA-exposed males</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No significant difference in egg production and fertilization of F1 and F2 females</span></span></span></li>
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No: F1 and F2</span></span></span></p>
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes: offspring of F2</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Female-biased sex ratio observed in both F1 and F2 adults</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">CRISPR/Cas9 mediated mutation of <em>eEF1A1b</em>; F1 sampled at 90, 120, 150 and 180 days after hatch</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant downregulation of key genes involving spermatogenesis</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Spermatogenesis arrested; reduced number of spermatogonia and spermatocytes</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced in vitro fertilization rate (5% vs 80% in WT) due to abnormal spermiogenesis</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult males exposed to two concentrations of bis-(2-ethylexhyl) phthalate (DEHP; 0.2 or 20 μg/L) for three weeks; 25 ng ethynylestradiol positive control</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Areas of spermatogonial and spermatid cysts were larger in fish exposed to 20 μg/L DEHP compared with controls</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Testicular area of spermatocyte cysts was lower in males exposed to 0.2 μg/L DEHP</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Testicular area occupied by spermatocytes was reduced in fish exposed to DEHP compared to controls, with a concomitant increase in the area occupied by spermatogonia</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in embryo production (up to 90%) observed in males treated with DEHP (0.2 and 20 μg/L)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Hatch rate of embryos significantly lower in DEHP-exposed males</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced expression of germ cell markers <em>vasa, dnd, piwil1 </em>and<em> amh</em> in mutants</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Deformed and apoptotic spermatogonia at 35 dpf found in mutants </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Lack of spermatozoa at adult stage </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Infertile under standard breeding despite being able to induce female egg laying (0% fertilization)</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Tudor domain-related proteins (Tdrds) have been demonstrated to be involved in spermatogenesis and Piwi-interacting RNA (piRNA) pathway </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Fish were exposed from 2 to 60 days post-hatch (dph) to nonylphenol (NP; 10, 30, or 100 μg/L nominal) or ethinylestradiol (EE2; 1, 10, or 100 ng/l nominal); reared until adulthood (120 dph) for breeding studies</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Majority of fish exposed to 10 ng/l EE lacked differentiated gonadal tissue (undeveloped gonads) at 60 dph</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">One fish at NP-30 μg/l and two fish at NP-100 μg/l were observed to have ovatestes at 60 dph</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish exposed to 10 ng/l of EE exhibited a significant reduction in the percent of viable eggs (clear vs opaque)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in hatch and swim-up success observed with EE2 and 100 μg NP/L</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Due to high mortality in the 100 ng/l EE group, insufficient fish were available for analyses</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Mature adult roach collected from both reference and river (effluent contaminated) sites during two consecutive spawning seasons; artificially induced to spawn in laboratory</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Volume of milt released from spermiating male fish significantly lower in the intersex fish than in the reference males</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Most fish that did not spermiate had testes that were clearly immature</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Fertilization rate significantly reduced when sperm from intersex males used to fertilize eggs collected from females</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Both proportion of fertilized embryos reaching eyed stage and hatching success decreased with increased feminization</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Embryo viability was determined after 24 h (fertilization success), at eyed stage and at swim-up stage (hatching success)</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult medaka exposed for 21 days to 29.3, 55.7, 116, 227, and 463 ng/L 17β-estradiol (E2)</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">In males exposed to 463 ng/l, a few oocytes were observed in testis, and testicular tissue almost completely replaced by connective tissue</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Accompanied by presence of macroscopic atrophy and degenerated spermatozoa and spermatocytes suggest a lack of spermatogenesis</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Total number of egg spawned and fertility significantly reduced at 463 ng/l E2 compared to the control</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Founder fish with originally <em>mlh1 </em>mutation was crossed out twice to WT fish of the TL line from which the founder was generated</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in weight of spermatids and spermatozoa); some spermatozoa were visible in testes of all mutant fish</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increased number and proportion of spermatogenic stages prior to spermatids compared to WT </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increase in apoptotic cells</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced fertilization rates under standard breeding conditions (0.4%)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Eggs fertilized from mutant sperm were malformed and and aneuploid</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Mlh1 is a member of DNA mismatch repair machinery and essential for stabilization of crossovers during first meiotic division </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3-month-old male fish exposed to 10 ug/L of DEHP for 3 months</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Semi-static exposure; half water renewed daily and whole water renewed weekly</span>; exposed males mated with WT females</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3-month-old male fish exposed to 30 ug/L of DEHP for 3 months</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3-month-old male fish exposed to 100 ug/L of DEHP for 3 months</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Percent of spermatocytes increased significantly by 27.4% </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease of 32.2% in spermatids</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization rate by 22% compared to the control</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Multi-generational study to 0.5, 5 and 50 ng/L ethynylestradiol (EE2) or 5 ng/L 17β-estradiol (E2)</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">None of the F<sub>1</sub> males exposed to 5 ng/L EE2 had normal testes; 43% had gonads not fully differentiated</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Time-related decrease in egg production and egg viability 14 hpf in F<sub>0</sub> generation at 50 ng/L EE2 and no survival of F<sub>1</sub> 100 hpf; no eggs produced after 10 d exposure </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Exposure to 5 ng/L EE2 in the F<sub>1</sub> caused a 56% reduction in fecundity and no survival past 14 hpf</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Proportion of nonviable eggs significantly higher for all treatments compared to control</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">Spermatogenesis is one of the most conserved biological processes from <em>Drosophila </em>to humans (Wu et al., 2016). The process itself is well understood and gametes produced from spermatogenesis are required for sexual reproduction.</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">When exposed to 50 mg DEHP kg-1 via intraperitoneal injection for 10 days, zebrafish experienced a reduction in the proportion of spermatozoa present compared to the control group. However, at this exposure concentration there was no effect on evidence for decrease in viable offspring. Whereas when exposed to 5000 mg of DEHP kg-1, there was a significantly lower proportion of spermatozoa and a significant decrease in fertilization success (Uren-Webster et al., 2010).</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">When exposed to DEHP for 3 months, zebrafish had a significant decrease in spermatids and increase in spermatocytes at the highest exposure concentration (100 ug/L) and no effect at the lowest exposure concentration (10 ug/L) (Ma et al. 2018)</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Targeted genetic disruption of <em>fdx1b</em> using a TALEN approach</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced sperm count compared to control (p=0.0097%)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Infertile under standard breeding despite being able to cause spawning of eggs (0% fertilization) </span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">fdx1b is an electron- providing cofactor for steroidogenic cytochrome P450</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">ENU mutagenesis screen to find mutations that lead to defects in gonadogenesis</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3 mutants focused on (<em>its, isa, imo</em>) </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Post meiotic germ cells absent at 3 months age (found aberrant germ cells instead)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Only spermatogonia and primary spermatocytes were present; no spermatids or sperm observed</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decreased fertilization rates in cells from mutant testes (<2% vs 41.9-65.8 in WT)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Only 1 mutant embryo survived at 1 dpf compared to nearly 100% in WT </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Loss of spermatozoa along with increase in primary spermatocytes compared to WT </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decrease in sperm count and sperm motility</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No viable offspring when mutants were crossed with any types of females</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Lethality of embryos via in vitro fertilization with WT females (before 1 dpf) </span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Mature fish exposed to </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">261, and 488 ng ethinylestradiol (EE2)/L for 21 d under flow-through conditions</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Testicular tissue composed of abnormally developed connective tissue, with only a few sper-matozoa and spermatocytes compared to control</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Upregulation of <em>amh</em> and <em>gsdf</em></span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced number of sperm</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduction in number of germ cells observed in AR mutant fish</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increased proportion of pre-spermatids sperm cells</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Small amount of mature spermatozoon still present in mutants</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Morphologically abnormal sperm (lacked tails and were degenerated)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced motility (12.5%) compared to control (51.5%)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Apoptotic spermatocytes likely due to destruction of Sertoli cells</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced in vivo fertilization rate (0.5%) vs control (94%) with WT females</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">PCI - present in seminal plasma; inhibitor of activated protein C and a variety of proteases</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult males exposed to 0.5 mg DEHP kg-1 (body weight) for 10 days via intraperitoneal injection</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">DEHP is phthalate which is a plasticizer in many mass-produced products</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult males exposed to 50 mg DEHP kg-1 for 10 days via intraperitoneal injection</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significantly lower proportion of spermatozoa and a significantly greater proportion of spermatocytes</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult males exposed to 5000 mg DEHP kg-1 for 10 days via intraperitoneal injection</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significantly lower proportion of spermatozoa and a significantly greater proportion of spermatocytes</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success of males, especially during the second 5-day period of exposure</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Post-meiotic development of elongating spermatids disrupted</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Abnormal morphology and degeneration of spermatids</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increased proportion of abnormal spermatids</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Downregulation of various spermatogenic markers</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">WT female mice coupled with homozygous mutant males did not produce any pups</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">BRD7 is a bromodomain gene that inhibits cell growth and cell cycle progression and is a co-factor for p53</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">BRD7 has high expression in mice testes </span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em><span style="color:black">mettl3</span></em><span style="color:black"> mutant fish generated using TALENs</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significantly increased proportions of spermatogonia (24.4% vs 7.5% in WT) and spermatocytes (56.1% vs 26.7% in WT) </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significantly decreased proportion of spermatozoa (10.4% vs 50.1% in WT)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Very little or no mature sperm</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decreased fertilization rate (48.8.% vs 91.4% in WT)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">8.1% of mutant male x WT female spawned successfully vs 94.4% in WT</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced number of spermatozoa compared to WT</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increased % of spermatocytes at leptotene and zygotene stages compared to WT </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Suggests arrest of spermatogenesis at zygotene stage; later stages rarely observed </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decreased fertilization rates (3% vs 94% in WT) under standard breeding conditions</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">E2f5 is a transcriptional repressor during cell-cycle progression</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">0.1 mg/L of DEHP for 6 months from larval stage</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Contained mostly spermatocytes (Sp) and spermatids (Sd) with few spermatozoa especially in this treatment</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fecundity compared to control (21.78 vs 29.89 eggs/f/d)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success (84.12 vs 94.21%)</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">MEHP - active metabolite of DEHP; </span>fertilization success defined as proportion of fertilized eggs</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">0.5 mg/L of DEHP for 6 months from larval stage</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Contained mostly Sp and Sd with few spermatozoa</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fecundity compared to control (20.44 vs 29.89 eggs/f/d)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success (81.61 vs 94.21%)</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">0.1 mg/L of MEHP for 6 months from larval stage</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Contained mostly Sp and Sd with few spermatozoa</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success vs control (87.46% vs 94.21%)</span></span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">0.5 mg/L of MEHP for 6 months from larval stage</span></span></span></p>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Contained mostly Sp and Sd with few spermatozoa</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success vs control (82.16% vs 94.21%)</span></span></span></li>
<strong>Uncertainties and Inconsistencies</strong>
<ul>
<li style="list-style-type:none"> </li>
<li><span style="font-size:11pt"><span style="font-size:12.0pt">When exposed to 10 and 100 ng/L of EE2 for 62 days leading to spawning, rainbow trout exhibited an increase in sperm density, concentration, and spermatocrit and decrease in GSI but overall there were no significant changes to spermatogenesis. Despite this, there was a decrease in viability of embryos (Schultz et al., 2003).</span></span></li>
<li><span style="font-size:11pt"><span style="font-size:12.0pt">Two-generation zebrafish study with 1 nM bisphenol A (BPA) showed a significant decrease in sperm density along with decreased sperm quality, however, no significant different in egg fertilization (Chen et al., 2015). </span></span></li>
<li><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:#212529">There are multiple other factors involved in producing viable offspring, including but not limited to </span>oocyte maturation and ovulation, development including successful organogenesis, and adequate nutrition.</span></span></li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">Empirical response-response data is very limited; thus, the response-response relationship has not yet been evaluated.</span></span></span></p>
<strong>Time-scale</strong>
<ul>
<li style="list-style-type:none"> </li>
<li><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">The duration of spermatogenesis in humans is reported to be 74 days (Griswold, M.D, 2016). Consequently, effects on spermatogenesis may not manifest as observable impacts on fertility until perhaps 74 days after impacts on spermatogenesis began. This may vary depending on the stage(s) of spermatogenesis that are impacted by the stressor.</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">The duration of the meiotic and spermiogenic phases in zebrafish is reported to be 6 days which means there could be a delay of at least 6 days before signs of impaired fertility and downstream effects may be detected (Leal et al., 2009).</span></span></span></li>
</ul>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">Feedforward/feedback loops haven’t been evaluated yet. However, given that that oocyte fertilization and production of viable offspring are external to the male it seems unlikely there would feedback that impacts spermatogenesis. </span></span></span></p>
<h4>References</h4>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Boulanger, G., Cibois, M., Viet, J., Fostier, A., Deschamps, S., Pastezeur, S., Massart, C., Gschloessl, B., Gautier-Courteille, C., & Paillard, L. (2015). Hypogonadism Associated with Cyp19a1 (Aromatase) Posttranscriptional Upregulation in Celf1 Knockout Mice. Molecular and cellular biology, 35(18), 3244–3253. https://doi.org/10.1128/MCB.00074-15</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Chen, J., Jiang, D., Tan, D., Fan, Z., Wei, Y., Li, M., & Wang, D. (2017). Heterozygous mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in male tilapia, Oreochromis niloticus. <em>Scientific reports</em>, <em>7</em>, 43733. https://doi.org/10.1038/srep43733</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Chen, J., Xiao, Y., Gai, Z., Li, R., Zhu, Z., Bai, C., Tanguay, R. L., Xu, X., Huang, C., & Dong, Q. (2015). Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects. Aquatic toxicology (Amsterdam, Netherlands), 169, 204–214. https://doi.org/10.1016/j.aquatox.2015.10.020</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Chen, J., Xiao, Y., Gai, Z., Li, R., Zhu, Z., Bai, C., Tanguay, R. L., Xu, X., Huang, C., & Dong, Q. (2015). Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects. <em>Aquatic toxicology (Amsterdam, Netherlands)</em>, <em>169</em>, 204–214. https://doi.org/10.1016/j.aquatox.2015.10.020</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Griswold M. D. (2016). Spermatogenesis: The Commitment to Meiosis. Physiological reviews, 96(1), 1–17. <a href="https://doi.org/10.1152/physrev.00013.2015" style="color:blue; text-decoration:underline"><span style="color:#337ab7">https://doi.org/10.1152/physrev.00013.2015</span></a></span></span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Leal, M. C., Feitsma, H., Cuppen, E., França, L. R., & Schulz, R. W. (2008). Completion of meiosis in male zebrafish (Danio rerio) despite lack of DNA mismatch repair gene mlh1. <em>Cell and tissue research</em>, <em>332</em>(1), 133–139. https://doi.org/10.1007/s00441-007-0550-z</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Ma, Yan-Bo, Jia, Pan-Pan, Junaid, Muhammad, Yang, Li, Lu, Chun-Jiao, & Pei, De-Sheng. (2018). Reproductive effects linked to DNA methylation in male zebrafish chronically exposed to environmentally relevant concentrations of di-(2-ethylhexyl) phthalate. Environmental Pollution (1987), 237, 1050-1061.</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Ma, Yan-Bo, Jia, Pan-Pan, Junaid, Muhammad, Yang, Li, Lu, Chun-Jiao, & Pei, De-Sheng. (2018). Reproductive effects linked to DNA methylation in male zebrafish chronically exposed to environmentally relevant concentrations of di-(2-ethylhexyl) phthalate. <em>Environmental Pollution </em>(1987), 237, 1050-1061.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Nash, J.P, Kime, D.E., Van der Ven, Leo T.M., Wester, P.W., Brion, F., Maack, G., Stahlschmidt-Allner, P., and Tyler, C.R., (2004). Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish. Environ Health Perspect 112(17):1725-1733.</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Saito, K., Siegfried, K. R., Nüsslein-Volhard, C., & Sakai, N. (2011). Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants. <em>Developmental dynamics : an official publication of the American Association of Anatomists</em>, <em>240</em>(7), 1779–1792. https://doi.org/10.1002/dvdy.22661</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Saju, J. M., Hossain, M. S., Liew, W. C., Pradhan, A., Thevasagayam, N. M., Tan, L., Anand, A., Olsson, P. E., & Orbán, L. (2018). Heat Shock Factor 5 Is Essential for Spermatogenesis in Zebrafish. <em>Cell reports</em>, <em>25</em>(12), 3252–3261.e4. https://doi.org/10.1016/j.celrep.2018.11.090</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Schultz, I. R., Skillman, A., Nicolas, J. M., Cyr, D. G., & Nagler, J. J. (2003). Short-term exposure to 17 alpha-ethynylestradiol decreases the fertility of sexually maturing male rainbow trout (Oncorhynchus mykiss). Environmental toxicology and chemistry, 22(6), 1272–1280.</span></span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="background-color:white"><span style="font-family:"Arial",sans-serif"><span style="color:#212121">Schulz, R. W., de França, L. R., Lareyre, J. J., Le Gac, F., Chiarini-Garcia, H., Nobrega, R. H., & Miura, T. (2010). Spermatogenesis in fish. <em>General and comparative endocrinology</em>, <em>165</em>(3), 390–411. https://doi.org/10.1016/j.ygcen.2009.02.013</span></span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Seki, M., Yokota, H., Matsubara, H., Tsuruda, Y., Maeda, M., Tadokoro, H. and Kobayashi, K. (2002). Effect of ethinylestradiol on the reproduction and induction of vitellogenin and testis-ova in medaka (Oryzias latipes). Environ. Toxicol. Chem. 21(8):1692-1698.</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Tang, H., Chen, Y., Wang, L., Yin, Y., Li, G., Guo, Y., Liu, Y., Lin, H., Cheng, C., & Liu, X. (2018). Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor. Biology of reproduction, 98(2), 227–238. https://doi.org/10.1093/biolre/iox165</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Tang, H., Chen, Y., Wang, L., Yin, Y., Li, G., Guo, Y., Liu, Y., Lin, H., Cheng, C., & Liu, X. (2018). Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor. <em>Biology of reproduction</em>, <em>98</em>(2), 227–238. https://doi.org/10.1093/biolre/iox165</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Uhrin, P., Dewerchin, M., Hilpert, M., Chrenek, P., Schöfer, C., Zechmeister-Machhart, M., Krönke, G., Vales, A., Carmeliet, P., Binder, B. R., & Geiger, M. (2000). Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility. <em>The Journal of clinical investigation</em>, <em>106</em>(12), 1531–1539. <a href="https://doi.org/10.1172/JCI10768" style="color:blue; text-decoration:underline">https://doi.org/10.1172/JCI10768</a></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Uren-Webster, Tamsyn M, Lewis, Ceri, Filby, Amy L, Paull, Gregory C, & Santos, Eduarda M. (2010). Mechanisms of toxicity of di(2-ethylhexyl) phthalate on the reproductive health of male zebrafish. Aquatic Toxicology, 99(3), 360-369.</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Uren-Webster, Tamsyn M, Lewis, Ceri, Filby, Amy L, Paull, Gregory C, & Santos, Eduarda M. (2010). Mechanisms of toxicity of di(2-ethylhexyl) phthalate on the reproductive health of male zebrafish. <em>Aquatic Toxicology</em>, <em>99</em>(3), 360-369.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Wang, H., Zhao, R., Guo, C., Jiang, S., Yang, J., Xu, Y., Liu, Y., Fan, L., Xiong, W., Ma, J., Peng, S., Zeng, Z., Zhou, Y., Li, X., Li, Z., Li, X., Schmitt, D. C., Tan, M., Li, G., & Zhou, M. (2016). Knockout of BRD7 results in impaired spermatogenesis and male infertility. <em>Scientific reports</em>, <em>6</em>, 21776. https://doi.org/10.1038/srep21776</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Wu, H., Sun, L., Wen, Y., Liu, Y., Yu, J., Mao, F., Wang, Y., Tong, C., Guo, X., Hu, Z., Sha, J., Liu, M., & Xia, L. (2016). Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans. Proceedings of the National Academy of Sciences of the United States of America, 113(15), 4134–4139. <a href="https://doi.org/10.1073/pnas.1513682113" style="color:blue; text-decoration:underline">https://doi.org/10.1073/pnas.1513682113</a></span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Xia, H., Zhong, C., Wu, X., Chen, J., Tao, B., Xia, X., Shi, M., Zhu, Z., Trudeau, V. L., & Hu, W. (2018). <em>Mettl3</em> Mutation Disrupts Gamete Maturation and Reduces Fertility in Zebrafish. <em>Genetics</em>, <em>208</em>(2), 729–743. https://doi.org/10.1534/genetics.117.300574</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Xie, H., Kang, Y., Wang, S., Zheng, P., Chen, Z., Roy, S., & Zhao, C. (2020). E2f5 is a versatile transcriptional activator required for spermatogenesis and multiciliated cell differentiation in zebrafish. <em>PLoS genetics</em>, <em>16</em>(3), e1008655. https://doi.org/10.1371/journal.pgen.1008655</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Ye, Ting, Kang, Mei, Huang, Qiansheng, Fang, Chao, Chen, Yajie, Shen, Heqing, & Dong, Sijun. (2014). Exposure to DEHP and MEHP from hatching to adulthood causes reproductive dysfunction and endocrine disruption in marine medaka (Oryzias melastigma). <em>Aquatic Toxicology, 146</em>, 115-126</span></span></span></span></span></span></p>
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<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2460">Relationship: 2460: Increased, Reactive oxygen species leads to Increased, LPO</a></h4>
<h4><a href="/relationships/2460">Relationship: 2460: Increase, ROS leads to Increase, LPO</a></h4>
<p>Considering the empirical domain of the evidence, the increased, reactive oxygen species leading to increased, lipid peroxidation is known to occur in fish and mammals, but, based on scientific reasoning, the biologically plausible domain of applicability can be eukaryotic organisms in general. It can be measured at any stage of life and in both male and female species.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Biological plausibility of this KER lies in the fact that reactive species, in excess, react and change macromolecules such as proteins, nucleic acids and lipids. Membrane lipids are particularly susceptible to damage by free radicals, as they are composed by unsaturated fatty acids (Su et al. 2019). Hence, increase in ROS production beyond antioxidant system defense capability of cells enables free circulation of molecules such as O2·−, HO·, H2O2, which removes electrons from membrane lipids and then triggers lipid peroxidation (Auten and Davis 2009; Su et al. 2019). </span></p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Analyses performed to support this relation show that KER3 is unchained by the three previously selected xenobiotics, as well as it takes place in a conserved way among species. Connection among the KEs is observed in both in vitro experimental models and in vivo systems, including fishes, birds and mammals.</span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">In cultures of rat hepatocytes, progressive ROS increase during 4 hours of treatment, triggered by DEM (5 mM), is followed by a continuous growth in levels of thiobarbituric acid reactive substances (TBARS), lipid peroxidation markers (Tirmenstein et al. 2000). This chemical depletes GSH content, leading to an augmentation of ROS levels and, consequently, to lipid peroxidation. In an in vivo model, 52 μM of DEM intraperitoneally injected in male Balb/c mice for two weeks caused a significant decrease in the GSH, increase in GSSG, ROS generation and increase in lipid peroxidation in testicles (Kalia and Bansal 2008).</span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">ATZ (46.4 µM) causes an increase of 48.97% of ROS and of 12.5% in MDA content in cultures of Sertoli-Germ cells from Wistar rats (25–28 days old), after, respectively, 3 and 24 h post-exposure. At a higher concentration (232 µM), these cells reach a maximum peak of ROS production after 6h of exposure, while MDA generation gets to the peak only after 24 h of treatment (Abarikwu, Pant, and Farombi 2012). In in vivo model, ATZ (38.5, 77 e 154 mg/Kg bw/day) led to a decrease in total antioxidant capacity (TAC) in a dose-dependent manner in male Sprague-Dawley rats of Specific Pathogen Free (SPF) ATZ-treated for 30 days. Which indirectly suggests increase in ROS levels – and increased malondialdehyde (MDA) content in 154 mg/Kg (Song et al. 2014). </span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">In relation to Hg, it was found that male young Wistar rats exposed to an initial dose of 4.6 μg/Kg of this metal (with following doses of 0.07 μg/Kg/day) displayed an increase in ROS levels, followed by an elevation of MDA content in testicles and epididymis of these rats 60 days post-exposure (Rizzetti et al. 2017). Other assays still carried out with male rats showed that the heavy metal induces oxidative stress with a single subcutaneous dose of 5 mg/Kg, by a substantial diminishment of activity of the main testicle antioxidant enzymes: SOD, CAT and GPX. Consequently, blood hydroperoxide and testicle MDA levels rose in a relevant way (El-Desoky et al. 2013).</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Furthermore, Hy-Line Brown laying hens fed with 4 experimental diets containing graded levels of Hg at 0.280, 3.325, 9.415, and 27.240 mg/Kg, respectively, for 10 weeks had GSH content significantly decreased in all Hg-treatment groups in ovaries, whilst SOD, CAT, GPX and glutathione reductase (GR) enzyme activities were significantly reduced, pointing to ROS accumulation. MDA content strongly increased in the 27.240-mg/Kg Hg group (Ma et al. 2018). </span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Hence, it can be deduced that, as in other adjacent relations evaluated, there is also evidence here that upstream KE is initially required in order to downstream KE take place, which reaffirms time concordance. Besides this, data enhance dose and incidence concordances for this KER.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Mechanisms involving lipid peroxidation, such as that one caused by ROS accumulation in cells, have been investigated for decades (Tirmenstein et al. 2000; Yin, Xu, and Porter 2011; Su et al. 2019). For this reason, there is much experimental data about response-response relationships or a growth of upstream KE in relation to downstream KE.</span></p>
<strong>Response-response relationship</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">This mechanism can be better understood through a process chain that consists of initiation, propagation and termination, as discussed by (Yin, Xu, and Porter 2011). In their review, these authors summarized a series of chemical reactions that develop during all this self-oxidation process and represent them in a schematic manner, as displayed in figure below.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Furthermore, although phospholipid oxidizability is lower, once their rate of diffusion in membranes is slower, the kinetics for this kind of reaction shown in figure follows the same law of velocity (steady-state rate) of homogeneous systems (equation below) (Yin, Xu, and Porter 2011). Oxygen consumption of the equation represents the rate of steady state, while rate of radical generation is defined by R<sub>i</sub>, the constant of propagation rate is expressed as k<sub>p</sub> and the termination rate constant for the reaction is called k<sub>t</sub>.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">For instance, empirical evidences show that rat hepatocytes begin ROS production after the first 30 minutes of DEM exposition (5 mM), growing linearly for all the remaining time, whereas the increase in products of lipid peroxidation (TBARS) starts only from the first hour of exposure (Tirmenstein et al. 2000).</span></p>
<td><span style="font-family:Arial,Helvetica,sans-serif">Auten and Davis 2009</span></td>
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</table>
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<h4>References</h4>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Su, Lian-Jiu, Jia-Hao Zhang, Hernando Gomez, Raghavan Murugan, Xing Hong, Dongxue Xu, Fan Jiang, and Zhi-Yong Peng. 2019. “Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis.” Oxidative Medicine and Cellular Longevity 2019 (October): 5080843.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Auten, Richard L., and Jonathan M. Davis. 2009. “Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details.” Pediatric Research 66 (2): 121–27.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Tirmenstein, M. A., F. A. Nicholls-Grzemski, J. G. Zhang, and M. W. Fariss. 2000. “Glutathione Depletion and the Production of Reactive Oxygen Species in Isolated Hepatocyte Suspensions.” Chemico-Biological Interactions 127 (3): 201–17.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Kalia, Sumiti, and M. P. Bansal. 2008. “Diethyl Maleate-Induced Oxidative Stress Leads to Testicular Germ Cell Apoptosis Involving Bax and Bcl-2.” Journal of Biochemical and Molecular Toxicology 22 (6): 371–81.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Abarikwu, S. O., E. O. Farombi, and A. B. Pant. 2011. “Biflavanone-Kolaviron Protects Human Dopaminergic SH-SY5Y Cells against Atrazine Induced Toxic Insult.” Toxicology in Vitro: An International Journal Published in Association with BIBRA 25 (4): 848–58.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Rizzetti, Danize Aparecida, Caroline Silveira Martinez, Alyne Goulart Escobar, Taiz Martins da Silva, José Antonio Uranga-Ocio, Franck Maciel Peçanha, Dalton Valentim Vassallo, Marta Miguel Castro, and Giulia Alessandra Wiggers. 2017. “Egg White-Derived Peptides Prevent Male Reproductive Dysfunction Induced by Mercury in Rats.” Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 100 (February): 253–64.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">El-Desoky, Gaber E., Samir A. Bashandy, Ibrahim M. Alhazza, Zeid A. Al-Othman, Mourad A. M. Aboul-Soud, and Kareem Yusuf. 2013. “Improvement of Mercuric Chloride-Induced Testis Injuries and Sperm Quality Deteriorations by Spirulina Platensis in Rats.” PloS One 8 (3): e59177.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Ma, Yan, Mingkun Zhu, Liping Miao, Xiaoyun Zhang, Xinyang Dong, and Xiaoting Zou. 2018. “Mercuric Chloride Induced Ovarian Oxidative Stress by Suppressing Nrf2-Keap1 Signal Pathway and Its Downstream Genes in Laying Hens.” Biological Trace Element Research 185 (1): 185–96.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Yin, Huiyong, Libin Xu, and Ned A. Porter. 2011. “Free Radical Lipid Peroxidation: Mechanisms and Analysis.” Chemical Reviews 111 (10): 5944–72.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Auten, Richard L., and Jonathan M. Davis. 2009. “Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details.” Pediatric Research 66 (2): 121–27.</span></p>