AOP-Wiki

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<div id="title">
<h2>AOP ID and Title:</h2>
<div class="title">AOP 521: Essential element imbalance leads to reproductive failure via oxidative stress</div>
Short Title: Essential element imbalance leads to reproductive failure via oxidative stress
</div>
<h2>Graphical Representation</h2>
<img src="https://training.aopwiki.org/system/dragonfly/production/2024/03/19/s6xdzezq6_AOP_521_Graphic.JPG" height="500" width="700" alt=""/>
<div id="authors">
<h2>Authors</h2>
<div>
Of the originating work:
Janaina da Silva (Department of General Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil)
Reggiani Vilela Gonçalves (Department of Animal Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil)
Fabiana Cristina Silveira Alves de Melo (Department of Animal Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil)
Mariáurea Matias Sarandy (Department of Animal Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil)
Sérgio Luis Pinto da Matta (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)
Of the content populated in the AOP-Wiki:
Travis Karschnik (General Dynamics Information Technology, Duluth, MN, USA.)
</div>
</div>
<div id="status">
<h2>Status</h2>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Author status</th>
<th scope="col">OECD status</th>
<th scope="col">OECD project</th>
<th scope="col">SAAOP status</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Under development: Not open for comment. Do not cite</td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
</div>
<div id="background">
~~</div>~~
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
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 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. Biological Trace Element Research, 199(7), 2663-2676. This publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages.
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.
 
</div>
<div id="development_strategy">
<h3>Strategy</h3>
<div>
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/ 
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, 
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.
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.
<img alt="" src="https://aopwiki.org/system/dragonfly/production/2024/04/05/5dwfnv7sfp_Citation_workflow_graphic.png" />
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.
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.
</div>
</div>
<div id="aop_summary">
<h2>Summary of the AOP</h2>
<h3>Events</h3>
<h3>Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sequence</th>
<th scope="col">Type</th>
<th scope="col">Event ID</th>
<th scope="col">Title</th>
<th scope="col">Short name</th>
</tr>
</thead>
<tbody>
<tr>
<td></td>
<td>MIE</td>
<td>2205</td>
<td><a href="/events/2205">Increased, essential element imbalance</a></td>
<td>Increased, essential element imbalance</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>KE</td>
<td>1115</td>
<td><a href="/events/1115">Increased, Reactive oxygen species</a></td>
<td>Increased, Reactive oxygen species</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1392</td>
<td><a href="/events/1392">Oxidative Stress </a></td>
<td>Oxidative Stress </td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1445</td>
<td><a href="/events/1445">Increased, Lipid peroxidation</a></td>
<td>Increased, LPO</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>2206</td>
<td><a href="/events/2206">Increased, histomorphological alteration of testis</a></td>
<td>Increased, histomorphological alteration of testis</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1758</td>
<td><a href="/events/1758">Impaired, Spermatogenesis</a></td>
<td>Impaired, Spermatogenesis</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>AO</td>
<td>2147</td>
<td><a href="/events/2147">Decreased, Viable Offspring</a></td>
<td>Decreased, Viable Offspring</td>
</tr>
</tbody>
</table>
</div>
<h3>Key Event Relationships</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Upstream Event</th>
<th scope="col">Relationship Type</th>
<th scope="col">Downstream Event</th>
<th scope="col">Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/relationships/3115">Increased, essential element imbalance</a></td>
<td>adjacent</td>
<td>Increased, Reactive oxygen species</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2009">Increased, Reactive oxygen species</a></td>
<td>adjacent</td>
<td>Oxidative Stress </td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/3116">Oxidative Stress </a></td>
<td>adjacent</td>
<td>Increased, Lipid peroxidation</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/3117">Increased, Lipid peroxidation</a></td>
<td>adjacent</td>
<td>Increased, histomorphological alteration of testis</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/3118">Increased, histomorphological alteration of testis</a></td>
<td>adjacent</td>
<td>Impaired, Spermatogenesis</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2937">Impaired, Spermatogenesis</a></td>
<td>adjacent</td>
<td>Decreased, Viable Offspring</td>
<td></td>
<td></td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2460">Increased, Reactive oxygen species</a></td>
<td>non-adjacent</td>
<td>Increased, Lipid peroxidation</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h3>Stressors</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Name</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Cadmium</td>
<td></td>
</tr>
<tr>
<td>Heavy metals (cadmium, lead, copper, iron, nickel)</td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="overall_assessment">
<h2>Overall Assessment of the AOP</h2>
<h3>Domain of Applicability</h3>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult, reproductively mature</td>
<td>High</td>
</tr>
<tr>
<td>Adult</td>
<td>High</td>
</tr>
<tr>
<td>Adults</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<div>
Taxonomic applicability: Murine models were the focus of the originating publication however the broader concepts likely apply to broader taxonomic groups.
Life stage applicability: The originating publication dealt with adult, reproductively mature organisms since the KEs were investigated in testis tissues and cells.
Sex applicability: Limited to male sex as constrained by testis.
In vitro data is used to support these domains.
</div>
</div>
<div id="considerations_for_potential_applicaitons">
</div>
<div id="references">
<h2>References</h2>
</div>
<div id="appendicies">
<h2>Appendix 1</h2>
<h3>List of MIEs in this AOP</h3>
<h4><a href="/events/2205">Event: 2205: Increased, essential element imbalance</a></h4>
<h5>Short Name: Increased, essential element imbalance</h5>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/521">Aop:521 - Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>testis</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult</td>
<td>High</td>
</tr>
<tr>
<td>All life stages</td>
<td></td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
<tr>
<td>Unspecific</td>
<td></td>
</tr>
</tbody>
</table>
</div>
Taxonomic applicability: AOP521 is focused on murine models but element imbalance and micromineral mimicry are not limited to this taxon.
Life stage applicability: AOP521 is focused on the adult life stage but element imbalance and micromineral mimicry are not limited to this life stage.
Sex applicability: AOP521 is focused on the Male sex but element imbalance and micromineral mimicry are not limited to this sex.
In vitro data is used to support these domains.
<h4>Key Event Description</h4>
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).
<h4>How it is Measured or Detected</h4>
Methods include X-ray emission, secondary ion emission and electron energy loss analysis. 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).
<h4>References</h4>
<div>Bindari, Y. R., Shrestha, S., Shrestha, N., & Gaire, T. N. (2013). Effects of nutrition on reproduction-A review. Advances in Applied Science Research, 4(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. Biological Trace Element Research, 199(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. Life sciences, 175, 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. Scanning Electron Microscopy, (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. Pedosphere, 24(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. Systems biology in reproductive medicine, 56(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. Biological trace element research, 174, 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. African journal of food science, 4(5), 200-222.</div>
<div> </div>
</div>
</div>
</div>
</div>
<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/1115">Event: 1115: Increased, Reactive oxygen species</a></h4>
<h5>Short Name: Increased, Reactive oxygen species</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>reactive oxygen species biosynthetic process</td>
<td>reactive oxygen species</td>
<td>increased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<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/213">Aop:213 - Inhibition of fatty acid beta oxidation leading to nonalcoholic steatohepatitis (NASH)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/303">Aop:303 - Frustrated phagocytosis-induced lung cancer</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/383">Aop:383 - Inhibition of Angiotensin-converting enzyme 2 leading to liver fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/382">Aop:382 - Angiotensin II type 1 receptor (AT1R) agonism leading to lung fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/384">Aop:384 - Hyperactivation of ACE/Ang-II/AT1R axis leading to chronic kidney disease </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/409">Aop:409 - Frustrated phagocytosis leads to malignant mesothelioma</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/413">Aop:413 - Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/416">Aop:416 - Aryl hydrocarbon receptor activation leading to lung cancer through IL-6 toxicity pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/418">Aop:418 - Aryl hydrocarbon receptor activation leading to impaired lung function through AHR-ARNT toxicity pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/386">Aop:386 - Deposition of ionizing energy leading to population decline via inhibition of photosynthesis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/387">Aop:387 - Deposition of ionising energy leading to population decline via mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/319">Aop:319 - Binding to ACE2 leading to lung fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/451">Aop:451 - Interaction with lung resident cell membrane components leads to lung cancer</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/476">Aop:476 - Adverse Outcome Pathways diagram related to PBDEs associated male reproductive toxicity</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/492">Aop:492 - Glutathione conjugation leading to reproductive dysfunction via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/497">Aop:497 - ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/505">Aop:505 - Reactive Oxygen Species (ROS) formation leads to cancer via inflammation pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/513">Aop:513 - Reactive Oxygen (ROS) formation leads to cancer via Peroxisome proliferation-activated receptor (PPAR) pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/521">Aop:521 - Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Cellular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>cell</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>organ</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Vertebrates</td>
<td>Vertebrates</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>human and other cells in culture</td>
<td>human and other cells in culture</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>crustaceans</td>
<td>Daphnia magna</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=35525" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Lemna minor</td>
<td>Lemna minor</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=4472" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
~~ROS is a normal constituent found in all organisms.~~
ROS is a normal constituent found in all organisms, lifestages, and sexes.
<h4>Key Event Description</h4>
~~Biological State: increased reactive oxygen species (ROS)~~
~~Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.~~
Biological State: increased reactive oxygen species (ROS)<br />
<strong>Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.
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).
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).
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.
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).
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].
<Free oxygen radicals>
<div>
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none">
<tbody>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px">
uperoxide
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px">
O2·-
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
hydroxyl radical
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
·OH
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
nitric oxide
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
NO·
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
organic radicals
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
R·
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
peroxyl radicals
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
ROO·
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
alkoxyl radicals
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
RO·
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
thiyl radicals
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
RS·
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
sulfonyl radicals
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
ROS·
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
thiyl peroxyl radicals
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
RSOO·
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
disulfides
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
RSSR
</td>
</tr>
</tbody>
</table>
</div>
<Non-radical ROS>
<div>
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none">
<tbody>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px">
hydrogen peroxide
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px">
H2O2
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
singlet oxygen
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
1O2
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
ozone/trioxygen
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
O3
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
organic hydroperoxides
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
ROOH
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
hypochlorite
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
ClO-
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
peroxynitrite
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
ONOO-
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
nitrosoperoxycarbonate anion
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
O=NOOCO2-
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
nitrocarbonate anion
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
O2NOCO2-
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
dinitrogen dioxide
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
N2O2
</td>
</tr>
<tr>
<td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
nitronium
</td>
<td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px">
NO2+
</td>
</tr>
<tr>
<td colspan="2" style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:580px">
highly reactive lipid- or carbohydrate-derived carbonyl compounds
</td>
</tr>
</tbody>
</table>
</div>
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 p47phox and p67phox. 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].
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.
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].
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].
<h4>How it is Measured or Detected</h4>
Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies.
Yuan, Yan, et al., (2013) described ROS monitoring by using H2-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H2-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.
Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013).
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> ~~
<div>
<Direct detection>
Many fluorescent compounds can be used to detect ROS, some of which are specific, and others are less specific.
・ROS can be detected by fluorescent probes such as p-methoxy-phenol derivative [Ashoka et al., 2020].
・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].
・ROS in the blood can be detected using superparamagnetic iron oxide nanoparticles (SPION)-based biosensor [Lee et al., 2020].
・Hydrogen peroxide (H2O2) can be detected with a colorimetric probe, which reacts with H2O2 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.
・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].
・Singlet oxygen can be measured by monitoring the bleaching of p-nitrosodimethylaniline at 440 nm using a spectrophotometer with imidazole as a selective acceptor of singlet oxygen [Onoue et al., 2014].
<Indirect Detection>
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.
</div>
<h4>References</h4>
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
Bedard, Karen, and Karl-Heinz Krause. 2007. “The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology.” Physiological Reviews 87 (1): 245–313.
Bisht, Shilpa, Muneeb Faiq, Madhuri Tolahunase, and Rima Dada. 2017. “Oxidative Stress and Male Infertility.” Nature Reviews. Urology 14 (8): 470–85.
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.
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.
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.
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.
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.
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.
Parrish, A. R. 2010. “2.27 - Hypoxia/Ischemia Signaling.” In Comprehensive Toxicology (Second Edition), edited by Charlene A. McQueen, 529–42. Oxford: Elsevier.
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.
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.
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.
<h4><a href="/events/1392">Event: 1392: Oxidative Stress </a></h4>
<h5>Short Name: Oxidative Stress </h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>oxidative stress</td>
<td></td>
<td>increased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/220">Aop:220 - Cyp2E1 Activation Leading to Liver Cancer</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/17">Aop:17 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress during brain development leads to impairment of learning and memory</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/284">Aop:284 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress leads to chronic kidney disease</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/411">Aop:411 - Oxidative stress Leading to Decreased Lung Function </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/424">Aop:424 - Oxidative stress Leading to Decreased Lung Function via CFTR dysfunction</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/425">Aop:425 - Oxidative Stress Leading to Decreased Lung Function via Decreased FOXJ1</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/429">Aop:429 - A cholesterol/glucose dysmetabolism initiated Tau-driven AOP toward memory loss (AO) in sporadic Alzheimer's Disease with plausible MIE's plug-ins for environmental neurotoxicants</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/452">Aop:452 - Adverse outcome pathway of PM-induced respiratory toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/464">Aop:464 - Calcium overload in dopaminergic neurons of the substantia nigra leading to parkinsonian motor deficits</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/470">Aop:470 - Deposition of energy leads to vascular remodeling</a></td>
<td>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/532">Aop:532 - CYP450 upregulation leads to Chronic kidney disease </a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Acetaminophen</td></tr>
<tr><td>Chloroform</td></tr>
<tr><td>furan</td></tr>
<tr><td>Platinum</td></tr>
<tr><td>Aluminum</td></tr>
<tr><td>Cadmium</td></tr>
<tr><td>Mercury</td></tr>
<tr><td>Uranium</td></tr>
<tr><td>Arsenic</td></tr>
<tr><td>Silver </td></tr>
<tr><td>Manganese</td></tr>
<tr><td>Nickel</td></tr>
<tr><td>Zinc</td></tr>
<tr><td>nanoparticles</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<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>~~
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.
~~~~
~~<h4>Aluminum</h4>~~
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.
~~~~
~~<h4>Cadmium</h4>~~
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).
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).
Jozefczak et al. (2015) treated Arabidopsis thaliana wildtype, cad2-1 mutant, and vtc1-1 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).
Andjelkovic et al. (2019) also found that renal lipid peroxidation was significantly increased in rats treated with 30 mg/kg of cadmium.
~~~~
~~<h4>Mercury</h4>~~
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.
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.
~~~~
~~<h4>Uranium</h4>~~
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.
Hao et al. (2014), 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).
~~~~
~~<h4>Arsenic</h4>~~
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).
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.
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.
~~~~
~~<h4>Silver </h4>~~
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.
~~~~
~~<h4>Manganese</h4>~~
Chtourou et al. (2012) investigated the effects of manganese treatment on rat kidneys. They found that manganese treatment caused significant increases in ROS production, lipid peroxidation, urinary H2O2 levels, and PCO production. They also found that intracellular GSH content was depleted in the treated group (Chtourou et al., 2012).
~~~~
~~<h4>Nickel</h4>~~
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).
~~~~
~~<h4>Zinc</h4>~~
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 μM or more for 8 weeks (Yeh et al., 2011).
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.
~~~~
~~<h4>nanoparticles</h4>~~
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).
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).
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.
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.
~~~~
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>rodents</td>
<td>rodents</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Homo sapiens</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic applicability: Occurrence of oxidative stress is not species specific.
Life stage applicability: Occurrence of oxidative stress is not life stage specific.
Sex applicability: Occurrence of oxidative stress is not sex specific.
Evidence for perturbation by prototypic stressor: 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).
<h4>Key Event Description</h4>
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.
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 (Pizzino et al., 2017; Sharifi-Rad et al., 2020; Jena et al., 2023).  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.
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).
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).
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).
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).
Sources of ROS Production
Direct Sources: 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).
Indirect Sources: 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 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 A2 (PLA2), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021).
<h4>How it is Measured or Detected</h4>
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. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed
<ul>
<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>
</ul>
Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include:
<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>
</ul>
<table border="1" cellpadding="1" cellspacing="1">
<tbody>
<tr>
<td>Assay Type & Measured Content</td>
<td>Description</td>
<td>Dose Range Studied</td>
<td>
Assay Characteristics (Length / Ease of use/Accuracy)
</td>
</tr>
<tr>
<td>
ROS Formation in the Mitochondria assay (Shaki et al., 2012)
</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>
Long/ Easy
High accuracy
</td>
</tr>
<tr>
<td>
Mitochondrial Antioxidant Content Assay Measuring GSH content
(Shaki et al., 2012)</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>
<td>
0, 50, 100, or 200 μM Uranyl Acetate
</td>
<td> </td>
</tr>
<tr>
<td>
H2O2 Production Assay Measuring H2O2 Production in isolated mitochondria
(Heyno et al., 2008)</td>
<td>“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 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>
<td>
0, 10, 30  μM Cd2+
2  μM
antimycin A</td>
<td> </td>
</tr>
<tr>
<td>
Flow Cytometry ROS & Cell Viability
(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 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)”</td>
<td> </td>
<td>
Strong/easy
medium</td>
</tr>
<tr>
<td>
DCFH-DA Assay Detection of hydrogen peroxide production (Yuan et al., 2016)
</td>
<td>
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.
</td>
<td>0-400 µM</td>
<td>
Long/ Easy
High accuracy
</td>
</tr>
<tr>
<td>
H2-DCF-DA Assay Detection of superoxide production (Thiebault et al., 2007)
</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>
Long/ Easy
High accuracy
</td>
</tr>
<tr>
<td>CM-H2DCFDA Assay</td>
<td>**Come back and explain the flow cytometry determination of oxidative stress from Pan et al. (2009)**</td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>
Direct Methods of Measurement
<table cellspacing="0" class="Table" style="border-collapse:collapse; width:623px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:141px">
Method of Measurement
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:151px">
References
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:255px">
Description
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:76px">
OECD-Approved Assay
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Chemiluminescence
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Lu, C. et al., 2006;
Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Spectrophotometry
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Direct or Spin Trapping-Based Electron Paramagnetic Resonance (EPR) Spectroscopy
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Nitroblue Tetrazolium Assay
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
The Nitroblue Tetrazolium assay is used to measure O2•– levels. O2•– reduces nitroblue tetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
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, hydrocyans can be oxidized by any ROS, and measured via fluorescence.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Amplex Red Assay
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
Fluorescence analysis to measure extramitochondrial or extracellular H2O2 levels. In the presence of horseradish peroxidase and H2O2, Amplex Red is oxidized to resorufin, a fluorescent molecule measurable by plate reader.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Dichlorodihydrofluorescein Diacetate (DCFH-DA)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
An indirect fluorescence analysis to measure intracellular H2O2 levels. H2O2 interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it to dichlorofluorescein (DCF), a fluorescent product.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
HyPer Probe
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
Fluorescent measurement of intracellular H2O2 levels. HyPer is a genetically encoded fluorescent sensor that can be used for in vivo and in situ imaging.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Cytochrome c Reduction Assay
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
The cytochrome c reduction assay is used to measure O2•– levels. O2•–  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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Proton-electron double-resonance imagine (PEDRI)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Glutathione (GSH) depletion
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Biesemann, N. et al., 2018)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
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">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</a>).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Thiobarbituric acid reactive substances (TBARS)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Griendling, K. K., et al., 2016)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Protein oxidation (carbonylation)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
(Azimzadeh et al., 2017; Azimzadeh etal., 2015; Ping et al., 2020)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
Can be determined with enzyme-linked immunosorbent assay (ELISA) or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">Seahorse XFp Analyzer   </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">Leung et al. 2018   </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">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).   </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">No </td>
</tr>
</tbody>
</table>
Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include:
<table cellspacing="0" class="Table" style="border-collapse:collapse; width:623px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:154px">
Method of Measurement
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:139px">
References
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:256px">
Description
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:75px">
OECD-Approved Assay
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">
Immunohistochemistry
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:139px">
(Amsen, D., de Visser, K. E., and Town, T., 2009)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:256px">
Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">
Quantitative polymerase chain reaction (qPCR)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:139px">
(Forlenza et al., 2012)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:256px">
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)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:46px; vertical-align:top; width:154px">
Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:46px; vertical-align:top; width:139px">
(Jackson, A. F. et al., 2014)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:46px; vertical-align:top; width:256px">
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
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:46px; vertical-align:top; width:75px">
No
</td>
</tr>
</tbody>
</table>
<h4>References</h4>
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/jisesa/ieab080</a>
Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, Antioxidants & Redox Signaling, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3400" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1089/ars.2010.3400</a>
Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, <a href="https://doi.org/10.1007/978-1-59745-447-6_5" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/978-1-59745-447-6_5</a>
Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, <a href="https://doi.org/10.1021/pr501141b" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1021/pr501141b</a>
Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, <a href="https://doi.org/10.1080/09553002.2017.1339332" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1080/09553002.2017.1339332</a>
Azzam, E. I. et al. (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, Cancer Letters, Vol. 327/1-2, Elsevier, Ireland, https://doi.org/10.1016/j.canlet.2011.12.012
Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, American Journal of Physiology - Cell Physiology, Vol. 318/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/ajpcell.00520.2019." style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/ajpcell.00520.2019.</a>
Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, Journal of ocular pharmacology and therapeutics, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, <a href="https://doi.org/10.1089/jop.2000.16.285.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a>
Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”, Scientific Reports, Vol. 8/1, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41598-018-27614-8" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/s41598-018-27614-8</a>.
Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in The Pathophysiologic Basis of Nuclear Medicine, Springer, New York, pp. 540-548
Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, Ophthalmic Research, Vol. 44, Karger International, Basel, <a href="https://doi.org/10.1159/000316476.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a>
Forlenza, M. et al. (2012), “The use of real-time quantitative PCR for the analysis of cytokine mRNA levels” in Cytokine Protocols, Springer, New York, <a href="https://doi.org/10.1007/978-1-61779-439-1_2" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/978-1-61779-439-1_2</a>
Forrester, S.J. et al. (2018), “Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology”, Physiological Reviews, Vol. 98/3, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00038.201" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/physrev.00038.201</a>
Foyer, C. H., A. V. Ruban, and G. Noctor (2017), “Viewing oxidative stress through the lens of oxidative signalling rather than damage”, Biochemical Journal, Vol. 474/6, Portland Press, England, https://doi.org/10.1042/BCJ20160814
Ganea, E. and J. J. Harding (2006), “Glutathione-related enzymes and the eye”, Current eye research, Vol. 31/1, Informa, London, <a href="https://doi.org/10.1080/02713680500477347.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/02713680500477347.</a>
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, Vol. 119/5, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/RES.0000000000000110" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/RES.0000000000000110</a>
Guo, C. et al. (2013), “Oxidative stress, mitochondrial damage and neurodegenerative diseases”, Neural regeneration research, Vol. 8/21, Publishing House of Neural Regeneration Research, China, <a href="https://doi.org/10.3969/j.issn.1673-5374.2013.21.009" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3969/j.issn.1673-5374.2013.21.009</a>
Hargreaves, M., and L. L. Spriet (2020), “Skeletal muscle energy metabolism during exercise.”, Nature Metabolism, Vol. 2, Nature Portfolio, London, <a href="https://doi.org/10.1038/s42255-020-0251-4" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/s42255-020-0251-4</a>
Hladik, D. and S. Tapio (2016), “Effects of ionizing radiation on the mammalian brain”, Mutation Research/Reviews in Mutation Research, Vol. 770, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.mrrev.2016.08.003" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.mrrev.2016.08.003</a>
Itoh, K., J. Mimura and M. Yamamoto (2010), “Discovery of the negative regulator of Nrf2, Keap1: a historical overview”, Antioxidants & Redox Signaling, Vol. 13/11, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3222" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1089/ars.2010.3222</a>
Jackson, A.F. et al. (2014), “Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan.”, Toxicology and Applied Pharmacology, Vol. 274/11, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.taap.2013.10.019" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.taap.2013.10.019</a>
Jacobsen, N.R. et al. (2008), “Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C60 fullerenes in the FE1-MutaTM Mouse lung epithelial cells”, Environmental and Molecular Mutagenesis, Vol. 49/6, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1002/em.20406" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1002/em.20406</a>
Karimi, N. et al. (2017), “Radioprotective effect of hesperidin on reducing oxidative stress in the lens tissue of rats”, International Journal of Pharmaceutical Investigation, Vol. 7/3, Phcog Net, Bengaluru, <a href="https://doi.org/10.4103/jphi.JPHI_60_17.%E2%80%AF" rel="noreferrer noopener" target="_blank">https://doi.org/10.4103/jphi.JPHI_60_17.</a>
Leung, D.T.H., and Chu, S. (2018), “Measurement of Oxidative Stress: Mitochondrial Function Using the Seahorse System” In: Murthi, P., Vaillancourt, C. (eds) Preeclampsia. Methods in Molecular Biology, vol 1710. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7498-6_22
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.trac.2006.07.007</a>
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1089/ars.2012.4641</a>
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1155/2020/3579143</a>
Pizzino, G. et al. (2017) “Oxidative Stress: Harms and Benefits for Human Health.” Oxidative medicine and cellular longevity, Vol. 2017: 8416763, Hindawi,  https://doi.org/10.1155/2017/8416763
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/physrev.00031.2007</a>
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/s41416-019-0651-y</a>
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1111/aos.13526</a>
Sharifi-Rad, M. et al. (2020), “Lifestyle, Oxidative Stress, and Antioxidants: Back and Forth in the Pathophysiology of Chronic Diseases.” Frontiers in physiology Vol. 11:694,  https://doi.org/10.3389/fphys.2020.00694
Snezhkina, A. V. et al. (2019), “ROS Generation and Antioxidant Defense Systems in Normal and Malignant Cells.” Oxidative medicine and cellular longevity Vol. 2019: 6175804, https://doi.org/10.1155/2019/6175804
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." style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/gerona/glt057.</a>
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/life11111269</a>
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.7150/ijbs.35460</a>
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/japplphysiol.01278.2007</a>.
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" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3892/ijmm.2019.4188</a>
<h4><a href="/events/1445">Event: 1445: Increased, Lipid peroxidation</a></h4>
<h5>Short Name: Increased, LPO</h5>
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>lipid oxidation</td>
<td>polyunsaturated fatty acid</td>
<td>increased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<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/532">Aop:532 - CYP450 upregulation leads to Chronic kidney disease </a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>fish</td>
<td>fish</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
ROS is a normal constituent found in all organisms, therefore, all organisms containing lipid membranes may be affected by lipid peroxidation.
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.
<h4>Key Event Description</h4>
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).
<h4>How it is Measured or Detected</h4>
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.
<h4>References</h4>
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
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.
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.
<h4><a href="/events/2206">Event: 2206: Increased, histomorphological alteration of testis</a></h4>
<h5>Short Name: Increased, histomorphological alteration of testis</h5>
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Abnormality of the testis</td>
<td>Testis</td>
<td>increased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/521">Aop:521 - Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>testis</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic applicability: AOP521 is focused on murine models but element imbalance and micromineral mimicry are not limited to this taxon.
Life stage applicability: Limited to adult organisms with reproductive tissues and structures.
Sex applicability: Limited to male organisms.
In vitro data is used to support these domains.
<h4>Key Event Description</h4>
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).
<h4>How it is Measured or Detected</h4>
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:
<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. Biological Trace Element Research, 199(7), 2663-2676.</div>
<h4><a href="/events/1758">Event: 1758: Impaired, Spermatogenesis</a></h4>
<h5>Short Name: Impaired, Spermatogenesis</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Abnormal spermatogenesis</td>
<td>Mature sperm cell</td>
<td>abnormal</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/323">Aop:323 - PPARalpha Agonism Leading to Decreased Viable Offspring via Decreased 11-Ketotestosterone</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/348">Aop:348 - Inhibition of 11β-Hydroxysteroid Dehydrogenase leading to decreased population trajectory </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/526">Aop:526 - Decreased, Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) leads to Impaired, Spermatogenesis</a></td>
<td>AdverseOutcome</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Flutamide</td></tr>
<tr><td>Vinclozolin</td></tr>
<tr><td>Bis(2-ethylhexyl) phthalate</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Organ</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>testis</td></tr>
</tbody>
</table>
</div>
~~<h3>Evidence for Perturbation by Stressor</h3>~~
~~<h4>Flutamide</h4>~~
~~Flutamide impairs spermatogenesis in adult male zebrafish (Yin et al., 2017)~~
~~Male fathead minnows exposed to flutamide show spermatocyte degredation and necrosis in their testis (Jensen et al., 2004)~~
~~~~
~~<h4>Vinclozolin</h4>~~
~~A review of androgen signaling in male fish cites several studies showing vinclozolin decreases sperm quality (Golshan et al., 2019)~~
~~~~
~~<h4>Bis(2-ethylhexyl) phthalate</h4>~~
~~A review of androgen signaling in male fish cites several studies showing DEHP decreases sperm quality (Golshan et al., 2019)~~
~~~~
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Vertebrates</td>
<td>Vertebrates</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult, reproductively mature</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic Applicability: The relevance for invertebrates has not been evaluated.
Life Stage Applicability: Only applicable for sexually mature adults
Sex Applicability: Only applicable to males
In vitro data is used to support these domains.
<h4>Key Event Description</h4>
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).
<h4>How it is Measured or Detected</h4>
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).
<h4>References</h4>
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>
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
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
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
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>
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
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
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>
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
<h3>List of Adverse Outcomes in this AOP</h3>
<h4><a href="/events/2147">Event: 2147: Decreased, Viable Offspring</a></h4>
<h5>Short Name: Decreased, Viable Offspring</h5>
~~<h4>Key Event Component</h4>~~
<h4>Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>sexual reproduction</td>
<td></td>
<td>decreased</td>
</tr>
<tr>
<td>viable offspring quantity</td>
<td></td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/323">Aop:323 - PPARalpha Agonism Leading to Decreased Viable Offspring via Decreased 11-Ketotestosterone</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/521">Aop:521 - Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>AdverseOutcome</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Individual</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult, reproductively mature</td>
<td>High</td>
</tr>
<tr>
<td>Adult</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td></td>
</tr>
</tbody>
</table>
</div>
Taxonomic applicability: Decrease in viable offspring may have relevance for species with sexual reproduction, including fish, mammals, amphibians, reptiles, birds, and invertebrates.
Life stage applicability: Decrease in viable offspring is relevant for reproductively mature individuals.
Sex applicability: Decrease in viable offspring can be measured for both males and females.
In vivo data is used to support these domains.
<h4>Key Event Description</h4>
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.
<h4>How it is Measured or Detected</h4>
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.
<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>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<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>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult, reproductively mature</td>
<td>High</td>
</tr>
<tr>
<td>Adult</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
</div>
<div>
<h4><a href="/relationships/2009">Relationship: 2009: Increased, Reactive oxygen species leads to Oxidative Stress </a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<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>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
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.
Sex: This key event relationship applies to both males and females.
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).
<h4>Key Event Relationship Description</h4>
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></p>
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.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
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></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).
Empirical Evidence
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none">
<tbody>
<tr>
<td style="background-color:#d0cece; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:138px">
Taxa
</td>
<td style="background-color:#d0cece; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:486px">
Support
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:138px">
Mammals
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:486px">
Deng et al. 2017; Schrinzi et al. 2017
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:138px">
Fish
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:486px">
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
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:138px">
Invertebrates
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:486px">
Browne et al. 2013; Jeong et al. 2016, 2017; Paul-Pont et al. 2016; Lei et al. 2018; Yu et al. 2018
</td>
</tr>
</tbody>
</table>
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.
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none">
<tbody>
<tr>
<td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:114px">
Species
</td>
<td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:72px">
Duration
</td>
<td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:120px">
Dose
</td>
<td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:48px">
Increased ROS?
</td>
<td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:66px">
Increased Oxidative Stress?
</td>
<td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:116px">
Summary
</td>
<td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:87px">
Citation
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Lab mice (Mus musculus)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
28 days
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Diet exposure of 0.01, 0.1, 0.5 mg/day of 5 and 20 um polystyrene microplastic particles.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Assumed1
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Deng et al. (2017)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Human (Homo sapiens)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
48 hours
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
In vitro exposure of 0.5, 1, 5, 10 mg/L fullerene soot, fullerol, graphene, cerium oxide, zirconium oxide, titanium oxide, aluminum oxide, silver nanoparticles, gold particles; in vitro exposure of 0.05, 0.1, 1, 10 mg/L polyethylene microspheres, polystyrene microspheres.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
Cerebral and epithelial human cell lines showed measured increased percent effect of ROS (as superoxide generated) with corresponding decreases in cell viability.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Schirinzi et al. (2017)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
7 days
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Aquatic exposure of 20, 200, 2000 ug/L of 5 and 20 um polystyrene microplastics.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Assumed1
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Lu et al. (2016)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Striped red mullet (Mullus surmuletus)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
NA
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Survey of wild fish with microplastic ingestion versus no microplastic ingestion.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Assumed1
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
Fish showed changes in enzyme levels responsible for eliminating ROS associated with microplastic ingestion, and associated proteins.  Increased glutathione S-transferase, superoxide dismutase, catalase, malondialdehyde, only glutathione S-transferase was statistically significant
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Alomar et al. (2017)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Zebrafish (Danio rerio)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
72 hours
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Aquatic exposure of 1 mg/L polystyrene microplastics (45 um) and nanoplastics (50 nm), aquatic exposure of 2, 20 ug/L positive control 17alpha-Ethinylestradiol, and mixture.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Assumed1
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
Larval fish showed changes in enzyme levels responsible for eliminating ROS.  Increased catalase, increased glutathione peroxidase, increased glutathione S-transferase.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Chen et al. (2017)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Zebrafish (Danio rerio)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
3 days
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Injection exposure of 5 mg/mL of 700 nm polystyrene particles
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Assumed1
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
Larva fish showed increased oxidative stress from gene ontology analysis.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Veneman et al. (2017)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
European Seabass (Dicentrarchus labrax)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
96 hours
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
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).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Barboza et al. (2018)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Sheepshead minnow (Cyprinodon variegatus)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
4 days
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Aquatic exposure of 50, 250 mg/L of 150-180 um, 300-355 um polyethylene microspheres
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
Adult fish showed increased ROS generation and corresponding changes in gene expression (increased catalase, increased superoxide dismutase).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Choi et al. (2018)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
24 hours
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
In vitro exposure of 100 mg/L of polyvinylchloride and polyethylene microplastics
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Assumed1
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
Fish head-kidney leucocytes showed increased gene expression of nuclear factor (nrf2), associated with oxidative stress, only statistically significant in S. aurata.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Espinosa et al. (2018)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Lugworms (Arenicola marina)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
10 days
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Aquatic exposure of nonylphenol (0.69-692.00 ug/g), phenanthrene (0.11-115.32 ug/g), PBDE (9.49-158.11 ug/g), triclosan (57.30-1097.87 ug/g) sorbed onto polyvinyl chloride, sand, or both.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
Lugworms showed decreased ability to respond to ROS by ferric reducing antioxidant power (FRAP) assay, statistically significant only with phenanthrene.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Browne et al. (2013)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Rotifer (Brachionus koreanus)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
24 hours
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Aquatic exposure of 10 ug/mL of 0.05, 0.5, 6 um diameter polystyrene microbeads.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
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).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Jeong et al. (2016)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Copepod (Paracyclopina nana)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
24 hours
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Aquatic exposure of 20 ug/mL of 0.05, 0.5, 6 um diameter polystyrene microbeads.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
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).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Jeong et al. (2017)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Mussel (Mytilus sp.)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
7 days
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
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.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Paul-Pont et al. (2016)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Nematode (Caenorhabditis elegans)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
2 day
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Environmental exposure of 5.0 mg/mL of microplastic particles (polyamides
(PA), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), and 0.1, 1.0, 5.0 um size polystyrene (PS)).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Assumed1
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
Larval (L2) nematodes showed increased glutathione S-transferase gene expression for all but polyamide (PA) exposure.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Lei et al. (2018)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:114px">
Crab (Eriocheir sinensis)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px">
21 days
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px">
Aquatic exposure of 40, 400, 4000, 40000 ug/L
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px">
Assumed1
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px">
Yes
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px">
Juvenile fish showed dose-dependent changes in hepatopancreas enzyme levels (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase), protein levels (glutathione, malondialdehyde) and gene expression (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase), as well as changes in MAPK signaling gene expression.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px">
Yu et al. (2018)
</td>
</tr>
</tbody>
</table>
1 Assumed: study selected stressor(s) known to elevate reactive oxygen species (ROS) levels, endpoints verified increased oxidative stress and disrupted pathway.
<h4>References</h4>
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
</div>
<div>
<h4><a href="/relationships/3116">Relationship: 3116: Oxidative Stress leads to Increased, LPO</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<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/532">CYP450 upregulation leads to Chronic kidney disease </a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<ul>
<li>Taxonomic applicability:  Most data was generated from human studies, bacteria, rat, or mice studies however ROS can affect all organisms containing lipid membranes and thus may be affected by lipid peroxidation due to oxidative stress.</li>
<li>Life stages: The domain of applicability for life stages is all life stages. </li>
<li> Sex applicability: The domain of applicability for sex is both males and females.</li>
<li>The biological plausibility for this key event relationship is strong.</li>
<li>The empirical evidence for this key event relationship is Strong.
</li>
</ul>
<h4>Key Event Relationship Description</h4>
The imbalance of reactive oxygen species to antioxidants, also known as oxidative stress, can result in lipid peroxidation. It has been well studied and established that radicals such as superoxide’s can interact with nucleophilic centers in the body like lipids in membrane bylayers. These lipids are composed of polyunsaturated fasts (PUFAs) like arachidonic acid which can become oxidized and lead to a chain reaction of oxidized lipids. More specifically, oxidation of PUFAS leads to the formation of another radical, a lipoperoxyl (LOO•), which, in turn, reacts with other lipids to yield not only another lipid radical but also a lipid hydroperoxide (LOOH). Although lipid hydroperoxides are unstable they offer some local adverse effects and can also create new radicals that decompose to secondary products with longer half-lives. These breakdown products include aldehydes such as acrolein and hexanal which can diffuse and react outside of its site of formation<cite> (Barrera et al., 2012)</cite>.  Antioxidants, such as vitamins or antioxidant enzymes, can react with lipid peroxy radicals to prevent further damage in the cell <cite>(Cooley et al., 2000)</cite>. In addition to this, antioxidants and antioxidant enzymes can also interact with reactive oxygen species to prevent ROS damage.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility for this key event relationship is strong: The relationship and mechanism between oxidative stress leading to lipid peroxidation is very well established and studied.
Empirical Evidence
The empirical evidence for this key event relationship is Strong. There are many studies linking oxidative stress to lipid peroxidation within both in vivo and in vitro research articles and most articles describe the mechanisms behind this KER.
Dose concordance:
An additional study that demonstrates great dose concordance in vivo is the administration of acrolein at concentration of 0-100 uM to human vascular endothelial cells. It was shown that exogenous acrolein can initiate oxidative stress and as a down stream result, lipid peroxidation through the creation of reactive oxygen species. A recent study in 2022 measured reactive oxygen species through two ROS specific dyes (MitoSox and DCH-DA) which revealed a significant increase in ROS at 50 uM of acrolein when compared to the control. In addition, the concentration of acrolein needed to induce a significant change in MDA measured cells was 100 uM thus proving that a lower dose was required to induce oxidative stress in human vascular endothelial cells than with lipid peroxidation via MDA measurement <cite>(Zhou et al, 2022).</cite>
One new dose concordance example follows the administration the ROS generating pharmaceutical- cyclophosphamide at concentrations of 0, 10, and 20 ug/ml to testicular Leydig cells. In this study it was seen that as the concentration of cyclophosphamide increased, the concentration of reactive oxygen species measured (via a ROS assay kit) very significantly increased at 10ug/ml. In addition, the concentration of MDA (measured via an MDA assay kit) followed a similar trend however only slightly significantly increased after 10 ug/ml of cyclophosphamide. This study showed that oxidative stress via reactive oxygen species required the same dose of stressor to illicit change in lipid peroxidation <cite>(Liao et al, 2024). </cite>
Another example of dose concordance with this KER is paraquat and hydrogen peroxide application to Vibrio cholerae <cite>(Abrashev et al., 2011)</cite>.  This study also demonstrated that the dose required for oxidative stress was less than/ equal to that needed to induce lipid peroxidation but through indirect markers. Cells were exposed to paraquat and hydrogen peroxide separately for one hour at concentrations of  0, 0.1, 0.3, 0.5, 1.0, 2.0, 3.0 mM. This resulted in increasing amounts of reactive oxygen species (specifically superoxide radicals and hydrogen peroxide) at 0.3 mM and higher. Similarly, lipid peroxidation and overall oxidative damage through protein carbonylation was measured at similar doses of 0, 0.1, 0.5, and 1 mM and showed the most change at 0.5 mM of paraquat <cite>(Abrashev et al., 2011, Rodríguez-García et al., 2020).</cite>
One final example of dose concordance is an in vitro study which exposed purified rat liver microsomal lipids to paraquat (a well studied oxidative stress inducer) in the presences of a NADPH-cytochrome c reductase. The cytochrome enzyme was included to interact with paraquat and include radicals which could then be measured against Malondialdehyde (MDA) concentrations as a result of lipid peroxidation. It was seen that as the concentration of paraquat increased from 0-0.0001 M the concentration of MDA also increased from 0.37 nmole/min/ml to 1.21 nmole/min/ml <cite>(Bus et al, 1976)</cite>. To make this study a perfect dose concordance experiment, including the concentration of reduced paraquat radicals would further the explanation of oxidative stress leading to lipid peroxidation.
Temporal concordance:
One example of temporal concordance regarding the relationship between oxidative stress and lipid peroxidation is the application of paraquat to mouse fibroblasts <cite>(Peter et al., 1991)</cite>. As the concentration of paraquat increased from 0-2.5 mM, and as a result radicals increased, the concentration of MDA also increased. MDA is a known metabolite of lipid peroxidation which was measured from 0-4 hours <cite>(Peter et al., 1991)</cite>. To make this a perfect temporal concordance experiment depiction, one would also need to include the measurement of an oxidative stress marker like reactive oxygen species production. This could be done by measuring superoxide radicals similar to the study done by Abrashev in 2011. This study with these modifications would be very fundamental in depicting oxidative stress preceding lipid peroxidation.
In addition, reactive oxygen species as a result of hyperglycemia in a study conducted in humans has been recommended to depict in vivo temporal concordance for future studies. Where an increase in glucose plasma levels overtime occurs before the occurrence of lipid peroxidation markers like MDA and 8-isoPGF2α <cite>(Ito et al., 2020)</cite>
Incidence concordance:
Much of the data found regarding incidence concordance was imperfect (just lacking population effects or 1/2 key event measurments) however one could expose a population of cells to an oxidative stress inducers like paraquat and measure the amount of lipid peroxidation and oxidative stress through ROS and oxidized lipid specific dyes with microscopy. Following this, one could measure the frequency of cells that show signs of oxidative stress (ex through ROS fluorescent dyes, which would show high fluorescence) and compare that to cells showing signs of lipid peroxidation (for instance a higher amount of membrane damage). A singular probe that can achieve this is Lipid Peroxidation Probe -BDP and has been used by multiple studies for similar experiments <cite>(Ma et al., 2023, Yang et al., 2023)</cite>. Hypothetically one should see a higher amount of cells conveying oxidative stress than cells conveying lipid peroxidation for incidence concordance to be true.
Uncertainties and Inconsistencies
The mechanism for this KER is very well understood and there is a high degree of concordance between many species, so far no large uncertainties or inconsistencies have been found.
<h4>Quantitative Understanding of the Linkage</h4>
Due to the fact that oxidative stress can originate from many factors, there are a vast amount of species that experience this phenomenon, and that there are multiple markers of lipid peroxidation, there is no set quantitative amount of oxidative stress that needs to occur before lipid peroxidation can be seen. However, it is widely accepted that continuous oxidative stress can result in lipid peroxidation.
Response-response relationship
N/A
Time-scale
N/A
Known modulating factors
<div>N/A
<table class="table table-bordered table-fullwidth">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>
</div>
Known Feedforward/Feedback loops influencing this KER
N/A
<h4>References</h4>
Abrashev R, Krumova E, Dishliska V, Eneva V, Engibarov S, Abrashev I & Angelova M, (2011) Differential Effect of Paraquat and Hydrogen Peroxide on the Oxidative Stress Response in Vibrio Cholerae Non O1 26/06, Biotechnology & Biotechnological Equipment, 25:sup1, 72-76,
Barrera G. (2012). Oxidative stress and lipid peroxidation products in cancer progression and therapy. ISRN oncology, 2012, 137289.
Bus J, Aust S, Gibson J. (1976). Paraquat Toxicity: Proposed Mechanism of Action Involving Lipid Peroxidation. Environmental health perspectives. 16. 139-46.
Cooley HM, Evans RE, Klaverkamp JF. (2000). Toxicology of dietary uranium in lake whitefish (Coregonus clupeaformis). Aquatic Toxicology. 48(4):495–515.
Ito F, Sono Y, Ito T. (2019) Measurement and Clinical Significance of Lipid Peroxidation as a Biomarker of Oxidative Stress: Oxidative Stress in Diabetes, Atherosclerosis, and Chronic Inflammation. Antioxidants (Basel).  Mar 25;8(3):72. doi: 10.3390/antiox8030072.
Liao S, Wei C, Wei G, Liang H, Peng F, Zhao L, Li Z, Liu C, Zhou Q, (2024) Cyclophosphamide activates ferroptosis-induced dysfunction of Leydig cells via SMAD2 pathway, Biology of Reproduction, (110) 5,1012-1024,
Ma D, Liu J, Wang L, Zhi X, Luo L, Zhao J, Qin Y., (2023) GSK-3β-dependent Nrf2 antioxidant response modulates ferroptosis of lens epithelial cells in age-related cataract, Free Radical Biology and Medicine, 204,161-176,
Peter B, Wartena M, Kampinga HH, Konings AW. (1991) Role of lipid peroxidation and DNA damage in paraquat toxicity and the interaction of paraquat with ionizing radiation. Biochem Pharmacol.  Feb 18;43(4):705-15.
Rodríguez-García A, García-Vicente R, Morales ML, Ortiz-Ruiz A, Martínez-López J, Linares M. (2020) Protein Carbonylation and Lipid Peroxidation in Hematological Malignancies. Antioxidants (Basel). Dec 1;9(12):1212
Yang H, Zhang X, Ding Y, Xiong H, Xiang S, Wang Y, Li H, Liu Z, He J, Tao Y, et al (2023). Elabela: Negative Regulation of Ferroptosis in Trophoblasts via the Ferritinophagy Pathway Implicated in the Pathogenesis of Preeclampsia. Cells.; 12(1):99.
Zhou Y, Xu H, Cheng K, Chen F, Zhou Q, Wang M. (2022) Acrolein evokes inflammation and autophagy-dependent apoptosis through oxidative stress in vascular endothelial cells and its protection by 6-C-(E-2-fluorostyryl)naringenin, Journal of Functional Foods, 98, 1756-4646,
</div>
<div>
<h4><a href="/relationships/3117">Relationship: 3117: Increased, LPO leads to Increased, histomorphological alteration of testis</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<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>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult, reproductively mature</td>
<td>High</td>
</tr>
<tr>
<td>Adult</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
</div>
<div>
<h4><a href="/relationships/3118">Relationship: 3118: Increased, histomorphological alteration of testis leads to Impaired, Spermatogenesis</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<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>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult, reproductively mature</td>
<td>High</td>
</tr>
<tr>
<td>Adult</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
</div>
<div>
<h4><a href="/relationships/2937">Relationship: 2937: Impaired, Spermatogenesis leads to Decreased, Viable Offspring</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/323">PPARalpha Agonism Leading to Decreased Viable Offspring via Decreased 11-Ketotestosterone</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Low</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>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>teleost fish</td>
<td>teleost fish</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=70862" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adult, reproductively mature</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic Applicability: Spermatogenesis is one of the most conserved biological processes from Drosophila 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.
Sex Applicability: Spermatogenesis is a male-specific process (Schulz et al., 2010, Tang et al., 2018, Wu et al., 2015 ). Thus, the present relationship is only relevant for males.
Life Stage Applicability: Spermatogenesis and reproduction are only relevant for sexually-mature adults.
<h4>Key Event Relationship Description</h4>
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.
<h4>Evidence Supporting this KER</h4>
Table 1A - Concordance table [authors A-N] (<a href="https://www.aopwiki.org/system/dragonfly/production/2023/09/29/4pdi2idto9_KER_2937_Concordance_Table.pdf">full table as PDF</a>)
<table cellspacing="0" class="Table" style="border-collapse:collapse; border:none; width:922px" summary="">
<tbody>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:101px">
Species
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:156px">
Experimental design
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:162px">
Evidence of Impaired Spermatogenesis (IS)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:144px">
Evidence of Viable Offspring, Decreased (VOD)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:78px">
IS observed?
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:78px">
VOD observed?
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:84px">
Citation
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:119px">
Notes
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Two generation exposure to 1nM BPA
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Significant decrease in sperm density of F1 and F2 males compared to control</li>
<li>Decreased sperm quality as measured by motility, velocity, ATP content and lipid peroxidation in F1 and F2 males</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>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</li>
<li>No significant difference in egg production and fertilization of F1 and F2 females</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
No: F1 and F2
Yes: offspring of F2
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Chen et al., 2015
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
Female-biased sex ratio observed in both F1 and F2 adults
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Tilapia (Oreochromis niloticus)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
CRISPR/Cas9 mediated mutation of eEF1A1b; F1 sampled at 90, 120, 150 and 180 days after hatch
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Significant downregulation of key genes involving spermatogenesis</li>
<li>Spermatogenesis arrested; reduced number of spermatogonia and spermatocytes</li>
<li>Altered morphology </li>
<li>Delayed spermatogenesis</li>
<li>Reduced motility</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Reduced in vitro fertilization rate (5% vs 80% in WT) due to abnormal spermiogenesis</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Chen et al., 2017
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
eEF1A1b - elongation factor
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
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
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Areas of spermatogonial and spermatid cysts were larger in fish exposed to 20 μg/L DEHP compared with controls</li>
<li>Testicular area of spermatocyte cysts was lower in males exposed to 0.2 μg/L DEHP</li>
<li>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</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Significant decrease in embryo production (up to 90%) observed in males treated with DEHP (0.2 and 20 μg/L)</li>
<li>Hatch rate of embryos significantly lower in DEHP-exposed males</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Corradetti et al., 2013
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
Reproductive performance evaluated with untreated females in clean water
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Targeted genetic disruption of tdrd12 through TALEN techniques
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Reduced expression of germ cell markers vasa, dnd, piwil1 and amh in mutants</li>
<li>Deformed and apoptotic spermatogonia at 35 dpf found in mutants </li>
<li>Lack of spermatozoa at adult stage </li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Infertile under standard breeding despite being able to induce female egg laying (0% fertilization)</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Dai et al., 2017
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
Tudor domain-related proteins (Tdrds) have been demonstrated to be involved in spermatogenesis and Piwi-interacting RNA (piRNA) pathway
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:none; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
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
</td>
<td style="border-bottom:none; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Majority of fish exposed to 10 ng/l EE lacked differentiated gonadal tissue (undeveloped gonads) at 60 dph</li>
<li>One fish at NP-30 μg/l and two fish at NP-100 μg/l were observed to have ovatestes at 60 dph</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Zebrafish exposed to 10 ng/l of EE exhibited a significant reduction in the percent of viable eggs (clear vs opaque)</li>
<li>Significant decrease in hatch and swim-up success observed with EE2 and 100 μg NP/L</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:none; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Hill and Janz, 2003
</td>
<td style="border-bottom:none; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
Due to high mortality in the 100 ng/l EE group, insufficient fish were available for analyses
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Roach
(Rutilus rutilus)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:156px">
Mature adult roach collected from both reference and river (effluent contaminated) sites during two consecutive spawning seasons; artificially induced to spawn in laboratory
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:162px">
<ul>
<li>Volume of milt released from spermiating male fish significantly lower in the intersex fish than in the reference males</li>
<li>Most fish that did not spermiate had testes that were clearly immature</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Fertilization rate significantly reduced when sperm from intersex males used to fertilize eggs collected from females</li>
<li>Both proportion of fertilized embryos reaching eyed stage and hatching success decreased with increased feminization</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:84px">
Jobling et al., 2002
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:119px">
Embryo viability was determined after 24 h (fertilization success), at eyed stage and at swim-up stage (hatching success)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Japanese medaka
(Oryzias latipes)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Adult medaka exposed for 21 days to 29.3, 55.7, 116, 227, and 463 ng/L 17β-estradiol (E2)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>In males exposed to 463 ng/l, a few oocytes were observed in testis, and testicular tissue almost completely replaced by connective tissue</li>
<li>Accompanied by presence of macroscopic atrophy and degenerated spermatozoa and spermatocytes suggest a lack of spermatogenesis</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Total number of egg spawned and fertility significantly reduced at 463 ng/l E2 compared to the control</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Kang et al., 2002
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Founder fish with originally mlh1 mutation was crossed out twice to WT fish of the TL line from which the founder was generated
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Significant decrease in weight of spermatids and spermatozoa); some spermatozoa were visible in testes of all mutant fish</li>
<li>Increased number and proportion of spermatogenic stages prior to spermatids compared to WT </li>
<li>Increase in apoptotic cells</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Reduced fertilization rates under standard breeding conditions (0.4%)</li>
<li>Eggs fertilized from mutant sperm were malformed and and aneuploid</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Leal et al., 2008
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
Mlh1 is a member of DNA mismatch repair machinery and essential for stabilization of crossovers during first meiotic division
</td>
</tr>
<tr>
<td rowspan="3" style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
3-month-old male fish exposed to 10 ug/L of DEHP for 3 months
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>No effect</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>No effect </li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
No
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
No
</td>
<td rowspan="3" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Ma et al., 2018
</td>
<td rowspan="3" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
Semi-static exposure; half water renewed daily and whole water renewed weekly; exposed males mated with WT females
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
3-month-old male fish exposed to 30 ug/L of DEHP for 3 months
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>No effect</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Concentration-dependent decrease in fertilization rate</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
No
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
3-month-old male fish exposed to 100 ug/L of DEHP for 3 months
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Percent of spermatocytes increased significantly by 27.4% </li>
<li>Significant decrease of 32.2% in spermatids</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Significant decrease in fertilization rate by 22% compared to the control</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Multi-generational study to 0.5, 5 and 50 ng/L ethynylestradiol (EE2) or 5 ng/L 17β-estradiol (E2)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>None of the F1 males exposed to 5 ng/L EE2 had normal testes; 43% had gonads not fully differentiated</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Time-related decrease in egg production and egg viability 14 hpf in F0 generation at 50 ng/L EE2 and no survival of F1 100 hpf; no eggs produced after 10 d exposure </li>
<li>Exposure to 5 ng/L EE2 in the F1 caused a 56% reduction in fecundity and no survival past 14 hpf</li>
<li>Proportion of nonviable eggs significantly higher for all treatments compared to control</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Nash et al., 2004
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
</td>
</tr>
</tbody>
</table>
Biological Plausibility
Spermatogenesis is one of the most conserved biological processes from Drosophila to humans (Wu et al., 2016). The process itself is well understood and gametes produced from spermatogenesis are required for sexual reproduction.
Empirical Evidence
Dose concordance
<ul>
<li>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).</li>
<li>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)</li>
</ul>
Table 1B - Concordance table [authors O-Z] (<a href="https://www.aopwiki.org/system/dragonfly/production/2023/09/29/4pdi2idto9_KER_2937_Concordance_Table.pdf">full table as PDF</a>)
<table cellspacing="0" class="Table" style="border-collapse:collapse; border:none; width:922px" summary="">
<tbody>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:101px">
Species
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:156px">
Experimental design
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:162px">
Evidence of Impaired Spermatogenesis (IS)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:144px">
Evidence of Viable Offspring, Decreased (VOD)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:78px">
IS observed?
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:78px">
VOD observed?
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:84px">
Citation
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:1px solid #a3a3a3; vertical-align:top; width:119px">
Notes
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Targeted genetic disruption of fdx1b using a TALEN approach
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Reduced sperm count compared to control (p=0.0097%)</li>
<li>Promale sox9a downregulated</li>
<li>Spermatogenic genes igf3 and insl3 downregulated</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Infertile under standard breeding despite being able to cause spawning of eggs (0% fertilization) </li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Oakes et al., 2019
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
fdx1b is an electron- providing cofactor for steroidogenic cytochrome P450
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<ul>
<li>ENU mutagenesis screen to find mutations that lead to defects in gonadogenesis</li>
<li>3 mutants focused on (its, isa, imo) </li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Post meiotic germ cells absent at 3 months age (found aberrant germ cells instead)</li>
<li>Only spermatogonia and primary spermatocytes were present; no spermatids or sperm observed</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Decreased fertilization rates in cells from mutant testes (<2% vs 41.9-65.8 in WT)</li>
<li>Only 1 mutant embryo survived at 1 dpf compared to nearly 100% in WT </li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Saito et al., 2011
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
ENU= N‐ethyl‐N‐nitrosourea
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
hsf5 mutants obtained by CRISPR/Cas9 technology targeting exon2
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Loss of spermatozoa along with increase in primary spermatocytes compared to WT </li>
<li>Decrease in sperm count and sperm motility</li>
<li>Altered morphology (microtubule arrangement, flagellar axoneme, sperm heads) </li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>No viable offspring when mutants were crossed with any types of females</li>
<li>Lethality of embryos via in vitro fertilization with WT females (before 1 dpf) </li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Saju et al., 2018
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
Heat shock protein 5
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Medaka
(Oryzias latipes)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Mature fish exposed to
32.6, 63.9, 116,
261, and 488 ng ethinylestradiol (EE2)/L for 21 d under ﬂow-through conditions
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Testicular tissue composed of abnormally developed connective tissue, with only a few sper-matozoa and spermatocytes compared to control</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Significant decrease in fecundity observed at 448 ng/L</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Seki et al., 2002
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish (Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<ul>
<li>ar mutant line generated using TALENs</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Upregulation of amh and gsdf</li>
<li>Downregulation igf3</li>
<li>Reduced number of sperm</li>
<li>Reduction in number of germ cells observed in AR mutant fish</li>
<li>Increased proportion of pre-spermatids sperm cells</li>
<li>Small amount of mature spermatozoon still present in mutants</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Reduced in vitro fertilization rate ≤ 20% with WT female</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Tang et al., 2018
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
Androgen receptor
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Mice
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<ul>
<li>mPCI deficient mice</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Morphologically abnormal sperm (lacked tails and were degenerated)</li>
<li>Reduced motility (12.5%) compared to control (51.5%)</li>
<li>Apoptotic spermatocytes likely due to destruction of Sertoli cells</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Reduced in vivo fertilization rate (0.5%) vs control (94%) with WT females</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Uhrin et al., 2000
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
<ul>
<li>PCI - present in seminal plasma; inhibitor of activated protein C and a variety of proteases</li>
</ul>
</td>
</tr>
<tr>
<td rowspan="3" style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Adult males exposed to 0.5 mg DEHP kg-1 (body weight) for 10 days via intraperitoneal injection
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>No effect</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>No effect</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
No
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
No
</td>
<td rowspan="3" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Uren-Webster et al., 2010
</td>
<td rowspan="3" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
DEHP is phthalate which is a plasticizer in many mass-produced products
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Adult males exposed to 50 mg DEHP kg-1 for 10 days via intraperitoneal injection
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Significantly lower proportion of spermatozoa and a significantly greater proportion of spermatocytes</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>No effect</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
No
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
Adult males exposed to 5000 mg DEHP kg-1 for 10 days via intraperitoneal injection
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Significantly lower proportion of spermatozoa and a significantly greater proportion of spermatocytes</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Significant decrease in fertilization success of males, especially during the second 5-day period of exposure</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Mice
(C57BL/6)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
BRD7-deficient mice
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Irregular head shape</li>
<li>Deformed acrosome</li>
<li>Post-meiotic development of elongating spermatids disrupted</li>
<li>Abnormal morphology and degeneration of spermatids</li>
<li>Increased proportion of abnormal spermatids</li>
<li>Downregulation of various spermatogenic markers</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>WT female mice coupled with homozygous mutant males did not produce any pups</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Wang et al., 2016
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
<ul>
<li>BRD7 is a bromodomain gene that inhibits cell growth and cell cycle progression and is a co-factor for p53</li>
<li>BRD7 has high expression in mice testes </li>
</ul>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
mettl3 mutant fish generated using TALENs
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Significantly increased proportions of spermatogonia (24.4% vs 7.5% in WT) and spermatocytes (56.1% vs 26.7% in WT) </li>
<li>Significantly decreased proportion of spermatozoa (10.4% vs 50.1% in WT)</li>
<li>Very little or no mature sperm</li>
<li>Sperm motility significantly reduced (average path velocity, curvilinear velocity, and straight-line velocity)</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Decreased fertilization rate (48.8.% vs 91.4% in WT)</li>
<li>8.1% of mutant male x WT female spawned successfully vs 94.4% in WT</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Xia et al., 2018
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
MEttl3 - multicomponent methyltransferase complex
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Zebrafish
(Danio rerio)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
CRISPR/Cas9 gene targeting of E2f5
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Reduced number of spermatozoa compared to WT</li>
<li>Increased % of spermatocytes at leptotene and zygotene stages compared to WT </li>
<li>Suggests arrest of spermatogenesis at zygotene stage; later stages rarely observed </li>
<li>Increased germ cell apoptosis</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Decreased fertilization rates (3% vs 94% in WT) under standard breeding conditions</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Xie et al., 2020
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
E2f5 is a transcriptional repressor during cell-cycle progression
</td>
</tr>
<tr>
<td rowspan="4" style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
Marine medaka (Oryzias melastigma)
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
0.1 mg/L of DEHP for 6 months from larval stage
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Contained mostly spermatocytes (Sp) and spermatids (Sd) with few spermatozoa especially in this treatment</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Significant decrease in fecundity compared to control (21.78 vs 29.89 eggs/f/d)</li>
<li>Significant decrease in fertilization success (84.12 vs 94.21%)</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td rowspan="4" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
Ye et al., 2014
</td>
<td rowspan="4" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
DEHP - phthalate
MEHP - active metabolite of DEHP; fertilization success defined as proportion of fertilized eggs
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
0.5 mg/L of DEHP for 6 months from larval stage
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Contained mostly Sp and Sd with few spermatozoa</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Significant decrease in fecundity compared to control (20.44 vs 29.89 eggs/f/d)</li>
<li>Significant decrease in fertilization success (81.61 vs 94.21%)</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
0.1 mg/L of MEHP for 6 months from larval stage
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Contained mostly Sp and Sd with few spermatozoa</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Significant decrease in fertilization success vs control (87.46% vs 94.21%)</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
0.5 mg/L of MEHP for 6 months from larval stage
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li>Contained mostly Sp and Sd with few spermatozoa</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li>Significant decrease in fertilization success vs control (82.16% vs 94.21%)</li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
Yes
</td>
</tr>
</tbody>
</table>
Uncertainties and Inconsistencies
<ul>
<li style="list-style-type:none"> </li>
<li>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).</li>
<li>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). </li>
<li>There are multiple other factors involved in producing viable offspring, including but not limited to oocyte maturation and ovulation, development including successful organogenesis, and adequate nutrition.</li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
Response-response relationship
Empirical response-response data is very limited; thus, the response-response relationship has not yet been evaluated.
Time-scale
<ul>
<li style="list-style-type:none"> </li>
<li>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.</li>
<li>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).</li>
</ul>
Known Feedforward/Feedback loops influencing this KER
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.
<h4>References</h4>
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
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. Scientific reports, 7, 43733. https://doi.org/10.1038/srep43733
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
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
Corradetti, B., Stronati, A., Tosti, L., Manicardi, G., Carnevali, O., and Bizzaro, D. (2013). Bis-(2-ethylexhyl) phthalate impairs spermatogenesis in zebrafish (Danio rerio). Reprod Biol. 13(3):195-202.
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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. Cell and tissue research, 332(1), 133–139. https://doi.org/10.1007/s00441-007-0550-z
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.
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</div>
<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>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/413">Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/492">Glutathione conjugation leading to reproductive dysfunction via oxidative stress</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/521">Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>non-adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>fish</td>
<td>fish</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Murinae gen. sp.</td>
<td>Murinae gen. sp.</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39108" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
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.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
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).
Empirical Evidence
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.
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).
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).
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).
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).
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.
<h4>Quantitative Understanding of the Linkage</h4>
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.
Response-response relationship
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.
<img alt="" src="https://pubs.acs.org/cms/10.1021/cr200084z/asset/images/medium/cr-2011-00084z_0012.gif" style="height:316px; width:500px" />
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 Ri, the constant of propagation rate is expressed as kp and the termination rate constant for the reaction is called kt.
-d[O] / dt = kp / (2kt)1/2. [L-H] . Ri1/2
Time-scale
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).
Known modulating factors
<div>
<table class="table table-bordered table-fullwidth">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td>antioxidant</td>
<td>vitamin E</td>
<td>prevents lipid peroxidation</td>
<td>Auten and Davis 2009</td>
</tr>
<tr>
<td>antioxidant</td>
<td>vitamin C</td>
<td>prevents lipid peroxidation</td>
<td>Auten and Davis 2009</td>
</tr>
</tbody>
</table>
</div>
<h4>References</h4>
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.
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.
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.
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.
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.
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.
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.
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.
Yin, Huiyong, Libin Xu, and Ned A. Porter. 2011. “Free Radical Lipid Peroxidation: Mechanisms and Analysis.” Chemical Reviews 111 (10): 5944–72.
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.
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