<td>Under development: Not open for comment. Do not cite</td>
<td></td>
<td></td>
<td></td>
</tr>
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</table>
</div>
</div>
<div id="coaches">
<h2>Coaches</h2>
<ul>
<li class="contributor" id="coach_109">
Shihori Tanabe
</li>
</ul>
</div>
<div id="abstract">
<h2>Abstract</h2>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This adverse outcome pathway (AOP 326) describes a linear route by which increased reactive oxygen species (ROS) can lead to decreased organismal growth through lipid peroxidation-mediated impairment of mitochondrial bioenergetics. In this AOP, increased ROS is treated operationally as the molecular initiating event because it represents the earliest common measurable redox perturbation shared by many chemical and non-chemical stressors within the broader ROS-growth AOP network. Increased ROS leads to oxidative stress, which promotes lipid peroxidation. Oxidative damage to membrane lipids, particularly polyunsaturated fatty acids in cellular and mitochondrial membranes, can alter membrane integrity, proton conductance, mitochondrial membrane potential, and respiratory efficiency. These effects reduce coupling of oxidative phosphorylation (OXPHOS), decrease the cellular ATP pool, impair energy-dependent cellular proliferation, and ultimately reduce organismal growth.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">AOP 326 reuses and connects established AOP-Wiki components from two important AOP contexts. The upstream oxidative stress component is associated with AOP 478, in which deposition of energy leads to oxidative stress through increased free radical generation, with subsequent oxidative molecular damage (AOP-Wiki, 2026a). This provides a curated AOP-Wiki context for the use of oxidative stress as a conserved hub KE downstream of free radical or ROS-producing stressors. The downstream bioenergetic and growth segment is directly associated with AOP 263, which causally links decreased coupling of OXPHOS to growth inhibition through ATP depletion and decreased cell proliferation and has been published in the OECD Series on Adverse Outcome Pathways (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). Thus, AOP 326 links an oxidative-stress/lipid-damage module to an OECD-endorsed bioenergetics-to-growth module. The AOP is relevant to environmental and human health contexts because ROS production, lipid peroxidation, mitochondrial ATP generation, cell proliferation, and organismal growth are broadly conserved in aerobic eukaryotes. </span> <span style="font-family:"Calibri",sans-serif">Empirical support is derived from studies in algae, daphnids, mollusks, fish, mammalian systems, and human cells exposed to redox-active chemicals, metals, pesticides, hypoxia-reoxygenation, radiation, and endogenous oxidative stressors. This AOP can support mechanistic interpretation of oxidative stress-mediated growth impairment, assay selection, chemical prioritization, integrated approaches to testing and assessment (IATA), and development of quantitative AOP approaches for mitochondrial and oxidative stress-related toxicity.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This project was funded by the Research Council of Norway (RCN), grant no. RCN-315929 “EXPECT: In silico and experimental screening platform for characterizing environmental impact of industry development in the Arctic” (https://www.niva.no/en/projects/expect), the European Partnership for the Assessment of Risks from Chemicals (PARC) through European Union’s Horizon Europe research and innovation programme (Grant Agreement No 101057014, and supported by the NIVA Computational Toxicology Program, NCTP (https://www.niva.no/en/featured-pages/nctp, grant. No. RCN-342628).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Artificial intelligence (AI) tools were used to support literature prioritization, review and AOP-Wiki page preparation in this work. AOP-helpFinder was used for automated literature mining, and ChatGPT (OpenAI) was used as an auxiliary tool for title and abstract screening, extraction of study metadata, and identification of potential weight-of-evidence indicators. AI-assisted outputs were used only to organize and prioritize information and were verified against the original sources by the authors before inclusion. Additional AI assistance was used for formatting, copy-editing, citation cross-checking, and harmonization of the AOP-Wiki pages. All scientific interpretations, weight-of-evidence judgments, final wording, and conclusions were determined and approved by the authors, who take full responsibility for the content and integrity of the work.</span></span></span></p>
</div>
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">ROS are continuously formed during aerobic metabolism and can also be generated in response to environmental stressors. At controlled levels, ROS participate in redox signaling, whereas excessive ROS can disturb redox homeostasis and initiate oxidative stress (Schieber and Chandel, 2014; Sies et al., 2017). Lipids are important targets of oxidative attack because membrane phospholipids, especially those containing polyunsaturated fatty acyl chains, can undergo radical-driven peroxidation. Lipid peroxidation generates lipid hydroperoxides and secondary reactive products, including malondialdehyde and 4-hydroxy-2-nonenal, which can alter membrane structure, propagate oxidative damage, and affect protein and organelle function (Ayala et al., 2014).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">AOP 326 was developed to represent the lipid peroxidation-driven linear route within the broader ROS-growth AOP network. This route was selected because lipid peroxidation is a well-established consequence of oxidative stress and because mitochondrial membranes are central determinants of OXPHOS coupling. Peroxidative modification of mitochondrial membrane lipids can alter membrane fluidity, proton leak, respiratory control, and mitochondrial membrane potential, providing a mechanistically coherent bridge from oxidative stress to impaired ATP production (Murphy, 2009; Nicholls and Ferguson, 2013; Ouillon et al., 2021). The downstream sequence from decreased coupling of OXPHOS to decreased ATP pool, decreased cell proliferation, and decreased growth is already represented in AOP 263 and provides the growth-relevant terminal module for AOP 326 (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).</span></span></span></p>
</div>
<div id="development_strategy">
<h3>Strategy</h3>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP 326 was developed using the principles described in OECD AOP guidance, including modular description of KEs and KERs, reuse of existing AOP-Wiki content where appropriate, evidence evaluation using biological plausibility, empirical support, essentiality, and quantitative understanding, and clear description of the biological domain of applicability (OECD, 2018, 2021). The purpose was to assemble a focused linear pathway from reusable AOP-Wiki elements rather than to create an isolated de novo pathway. This is particularly important because AOP 326 is one branch of the broader ROS-growth AOP network and because its KEs and KERs overlap with oxidative stress, mitochondrial dysfunction, energy metabolism, cell proliferation, and growth-related AOPs.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">Reuse of existing AOP-Wiki content was considered at the beginning of development. AOP 478 was reviewed because it provides an AOP-Wiki precedent for oxidative stress as a central KE downstream of free radical generation, and because it describes oxidative stress as a pathway by which energy deposition can damage biological molecules (AOP-Wiki, 2026a). Although AOP 478 is not a lipid peroxidation-to-growth AOP, its use of KE 1392 (Increase, Oxidative stress) and its oxidative damage context support the upstream portion of AOP 326. AOP 263 was reviewed because it provides the directly relevant downstream module consisting of KE 1446 (Decrease, Coupling of OXPHOS), KE 1771 (Decrease, ATP pool), KE 1821 (Decrease, Cell proliferation), and AO 1521 (Decrease, Growth), together with KERs 2203, 2204, and 2205 (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). In AOP 326, the novel linking logic is therefore the connection of ROS-driven oxidative stress and lipid peroxidation to the existing OXPHOS-ATP-cell proliferation-growth module.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">The evidence base was assembled through a structured AI-human hybrid workflow. Search terms were first developed for the events in the pathway, including KE names, synonyms, endpoint names, assay terms, taxa, and species. AOP-helpFinder was then used to search PubMed for co-occurrence of KE-related terms, following approaches previously described for literature mining in support of AOP development (Carvaillo et al., 2019; Jornod et al., 2022). The exported AOP-helpFinder results included PMIDs, titles, abstracts, and matched KE terms. Overlap analysis was applied to remove redundant hits and filter literature that was unrelated to the taxa or biological systems considered relevant to this AOP.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">A large language model (LLM) was then used as an auxiliary screening and structuring tool. During abstract pre-screening, the LLM was used to extract study metadata such as stressor, species, biological system, dose or concentration, and exposure time; to identify whether a study provided evidence for biological plausibility, empirical support, essentiality, or quantitative understanding; and to flag indicators of dose-response, temporal, or incidence concordance. Studies were provisionally classified as high, medium, or low priority. High-priority studies were retrieved for full-text review, medium-priority studies were reserved for supporting evidence, and low-priority studies were documented as low relevance. For studies passing the abstract screen, the LLM was used to assist full-text review by organizing information relevant to the KER evidence table. All LLM outputs were checked manually against the full text by expert reviewers before any evidence was accepted.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">The final phase consisted of manual expert curation. Experts verified the LLM-assisted extractions, populated KER evidence tables with methods, endpoints, results, weight-of-evidence categories, and references, and made final calls for biological plausibility, empirical support, essentiality, and evidence gaps. Targeted manual searches were also used to fill gaps identified during evidence curation, especially for the lipid peroxidation to OXPHOS coupling relationship and the downstream AOP 263 KERs. Searches focused on combinations of terms such as reactive oxygen species, oxidative stress, lipid peroxidation, malondialdehyde, 4-hydroxynonenal, mitochondrial membrane potential, proton leak, oxidative phosphorylation, ATP depletion, cell proliferation, growth inhibition, paraquat, copper, cadmium, hypoxia-reoxygenation, Daphnia, algae, bivalves, fish, and AOP. Studies were prioritized when they measured two or more KEs in the same biological system, reported exposure dose or concentration and time, or provided evidence relevant to dose-response, temporal, or incidence concordance.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The overall weight of evidence supporting AOP 326 is considered moderate to high. Biological plausibility is high for all six KERs in the pathway, reflecting well-established mechanistic connections between ROS, oxidative stress, lipid peroxidation, mitochondrial membrane integrity, OXPHOS coupling, ATP production, cell proliferation, and growth. The downstream bioenergetic module from decreased coupling of OXPHOS through decreased ATP pool and decreased cell proliferation to decreased growth (KERs 2203, 2204, 2205) is directly reused from OECD-endorsed AOP 263, which was published in the OECD Series on Adverse Outcome Pathways and has independently established high to moderate WoE for these relationships (OECD, 2022; Song and Villeneuve, 2021). The upstream oxidative stress-lipid peroxidation module is supported by high empirical concordance across algae, crustaceans, bivalves, fish, and mammalian systems. The novel linking relationship from lipid peroxidation to decreased OXPHOS coupling (KER 1599) is rated moderate in empirical support, as studies measuring both endpoints concurrently in the same experimental system are less common, although mechanistic evidence for cardiolipin oxidation and inner mitochondrial membrane integrity is strong. Essentiality is rated moderate to high, with the strongest support for the OXPHOS-to-ATP segment shared with AOP 263. Quantitative understanding is highest for the AOP 263-derived downstream module and low to moderate for the upstream lipid peroxidation-to-OXPHOS segment. The main uncertainties are the directionality and quantitative strength of the lipid peroxidation-to-OXPHOS relationship, given that mitochondrial dysfunction can also promote lipid peroxidation, and the potential for compensatory glycolytic ATP production to buffer depletion. AOP 326 is suitable for mechanistic interpretation, IATA development, and chemical prioritisation for oxidative stress-mediated growth impairment affecting mitochondrial energy metabolism (OECD, 2018; OECD, 2022; Becker et al., 2015).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The domain of applicability for AOP 326 is broad across aerobic eukaryotic organisms in which ROS generation, oxidative stress responses, lipid peroxidation, mitochondrial oxidative phosphorylation, ATP-dependent cell proliferation, and growth are biologically relevant. The AOP is most applicable to taxa and life stages in which growth depends substantially on cell proliferation and energy supply, including algae, developing aquatic organisms, juvenile fish, and proliferating mammalian or human cells. It is also relevant to adult organisms when growth, regeneration, tissue condition, or organismal size is influenced by mitochondrial energy metabolism.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The stressor domain is also broad and includes direct ROS generators, redox-cycling chemicals, metals, pesticides, mitochondrial toxicants, hypoxia-reoxygenation, and radiation. Because the MIE is defined operationally as increased ROS rather than as a chemical-specific interaction, AOP 326 should be applied to stressors that can be shown to increase ROS or oxidative stress and to produce evidence consistent with lipid peroxidation and mitochondrial bioenergetic impairment. Environmental factors such as temperature, oxygen availability, diet, lipid composition, nutrient status, and antioxidant capacity may modulate the pathway by altering ROS production, lipid susceptibility to peroxidation, mitochondrial coupling, or growth rate.</span></span></span></p>
<h3>Essentiality of the Key Events</h3>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Essentiality is evaluated for the overall AOP based on whether preventing or modifying upstream KEs changes downstream KEs or the AO. Direct essentiality evidence is strongest for the AOP 263 downstream module, where removal of mitochondrial uncouplers or restoration of coupling can restore mitochondrial membrane potential and ATP production. Essentiality for lipid peroxidation is biologically plausible and supported by intervention and association studies, but direct experiments showing that blocking lipid peroxidation prevents all downstream events are less common.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">ROS scavenging or antioxidant interventions frequently attenuate oxidative stress and downstream lipid peroxidation in oxidative stress models. ROS is also used as a common early perturbation in the broader ROS-growth AOP network (Schieber and Chandel, 2014; Sies et al., 2017).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Indirect (stop/attenuation): antioxidant and ROS-scavenger pre-treatment reduces oxidative stress and downstream damage across oxidative-stress models (Schieber and Chandel, 2014; Sies et al., 2017). No selective single-source ROS knock-out is available.</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">ROS also participate in normal signaling; increased ROS does not always progress to adversity if antioxidant and repair systems compensate.</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Oxidative stress is required for lipid peroxidation when ROS production exceeds antioxidant buffering. AOP 478 supports oxidative stress as a central KE downstream of free radical generation (AOP-Wiki, 2026a).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Indirect: modulation of antioxidant capacity alters progression to oxidative macromolecular damage; oxidative stress is the curated hub KE in endorsed AOP 478 (AOP-Wiki, 2026a; Carrothers et al., 2025).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Oxidative stress can be measured using multiple indirect biomarkers; equivalence across methods is not always clear.</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Lipid peroxidation can alter membrane properties and generate reactive aldehydes that affect mitochondrial function (Ayala et al., 2014). Dietary PUFA studies in Daphnia show higher lipid peroxidation with lower mitochondrial membrane potential (Moore et al., 2023), supporting a causal role in mitochondrial impairment.</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Indirect: antioxidant intervention attenuates lipid peroxidation in oxidative-stress models; direct block-and-rescue isolating LPO from other oxidative damage is uncommon (Murphy, 2009; Ouillon et al., 2021).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Direct blocking experiments are limited; lipid peroxidation may be both a cause and consequence of mitochondrial dysfunction.</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">This KE is reused from AOP 263. Evidence from AOP 263 supports essentiality because removal of uncouplers or restoration of coupling can recover mitochondrial membrane potential and ATP levels, reducing downstream impairment (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Direct (rescue): removal of uncouplers or restoration of coupling recovers mitochondrial membrane potential and ATP in the endorsed AOP 263 module (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Mild uncoupling can sometimes reduce ROS generation and may be adaptive; severity and duration determine progression.</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Event 1771: ATP pool, decreased</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">ATP depletion is a direct consequence of impaired OXPHOS coupling and is associated with reduced cell proliferation and cytotoxicity in multiple systems. AOP 263 identifies ATP depletion as an intermediate KE linking OXPHOS uncoupling to reduced proliferation (AOP-Wiki, 2026b; OECD, 2022).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Cells may compensate through glycolysis or other energy pathways; total ATP may recover transiently depending on exposure scenario.</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Growth depends on accumulation of cell number and biomass. AOP 263 provides evidence that decreased cell proliferation links ATP depletion to decreased growth (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Indirect: proliferation deficit links bioenergetic/genotoxic upstream to growth; reused from endorsed AOP 263 with KER 2205 (AOP-Wiki, 2026d; Conlon and Raff, 1999; OECD, 2022; Song and Villeneuve, 2021).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Growth can be influenced by cell size, metabolism, development, nutrient status, and cell death, not proliferation alone.</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Growth is the adverse outcome and is regulatory relevant across algae, aquatic invertebrate, fish, amphibian, and plant test systems. AOP 263 provides precedent for using decreased growth as the AO in a mitochondrial bioenergetics AOP (OECD, 2022; Song and Villeneuve, 2021).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">As the adverse outcome, essentiality is assessed for upstream KEs; AOP 263 provides precedent for decreased growth as an AO downstream of these modules (OECD, 2022; Song and Villeneuve, 2021).</span></span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Growth is an integrative endpoint and can arise through multiple independent or interacting mechanisms.</span></span></span></span></p>
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<p> </p>
<h3>Weight of Evidence Summary</h3>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Evidence assessment is organized by KER. Calls follow OECD weight-of-evidence considerations for biological plausibility, empirical support, and quantitative understanding (OECD, 2018, 2021).</span></span></span></p>
<p> </p>
<h3><span style="font-size:18px"><span style="font-family:Calibri,sans-serif"><span style="color:#4f81bd">Biological plausibility of KERs</span></span></span></h3>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The relationship is mechanistically established because oxidative stress reflects an imbalance between oxidant production and antioxidant capacity, and ROS are primary oxidant species in cellular redox biology (Schieber and Chandel, 2014; Sies et al., 2017). AOP 478 provides a curated AOP-Wiki context for oxidative stress downstream of free radical generation (AOP-Wiki, 2026a).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Free radicals and other ROS can initiate peroxidation of polyunsaturated fatty acids in membranes, generating lipid hydroperoxides and reactive aldehydes such as MDA and 4-HNE (Ayala et al., 2014; Sies et al., 2017).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Mitochondrial coupling depends on the integrity and composition of the inner mitochondrial membrane. Lipid peroxidation can disrupt membrane properties, promote proton leak, alter membrane potential, and impair respiratory control (Murphy, 2009; Nicholls and Ferguson, 2013; Ouillon et al., 2021).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Relationship 2203: decreased coupling of OXPHOS leads to decreased ATP pool</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This relationship is directly reused from AOP 263. OXPHOS coupling is a major determinant of ATP production in aerobic eukaryotic cells; reduced coupling lowers ATP synthesis efficiency (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Relationship 2204: decreased ATP pool leads to decreased cell proliferation</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This relationship is reused from AOP 263. Cell proliferation requires ATP for DNA replication, mitosis, biosynthesis, and maintenance of cellular processes; ATP depletion therefore plausibly reduces proliferation (AOP-Wiki, 2026b; OECD, 2022; Bonora et al., 2012; Song and Villeneuve, 2021).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This relationship is reused from AOP 263. Organismal, tissue, and population growth require accumulation of cells and biomass; sustained reduction in proliferation is therefore expected to reduce growth (AOP-Wiki, 2026b; Conlon and Raff, 1999; OECD, 2022).</span></span></span></p>
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<p> </p>
<h3><span style="font-size:18px"><span style="font-family:Calibri,sans-serif"><span style="color:#4f81bd">Empirical support for KERs</span></span></span></h3>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Multiple studies demonstrate concordance between ROS or ROS-producing stressors and oxidative stress markers. Paraquat increased ROS and antioxidant enzyme responses in Chlorella vulgaris (Qian et al., 2009). Paraquat also induced oxidative stress responses and lipid peroxidation in Daphnia magna (Barata et al., 2005).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">ROS is often measured indirectly and may be transient; oxidative stress biomarkers vary by assay and tissue.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Oxidative stress and lipid peroxidation are often observed together. Copper increased antioxidant enzyme activity and MDA/TBARS in freshwater green microalgae (Knauert and Knauer, 2008). Paraquat and other redox-cycling stressors induced lipid peroxidation in algae and Daphnia (Barata et al., 2005; Esperanza et al., 2015; Qian et al., 2009). In the aquatic macrophyte Lemna minor, gamma radiation and the respiratory uncoupler 3,5-dichlorophenol induced a concordant oxidative stress to lipid peroxidation sequence preceding growth inhibition (Xie et al., 2018; Xie et al., 2019; Xie et al., 2022).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Lipid peroxidation can occur at different times than enzyme responses, and MDA/TBARS assays have specificity limitations.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Dietary PUFA manipulation in Daphnia showed higher lipid peroxidation associated with lower mitochondrial membrane potential (Moore et al., 2023). Cyclic hypoxia in Mya arenaria increased proton leak and reduced OXPHOS coupling efficiency, consistent with oxidative lipid and membrane damage effects on mitochondrial coupling (Ouillon et al., 2021). In Lemna minor, gamma radiation and 3,5-dichlorophenol reduced mitochondrial membrane potential downstream of lipid peroxidation, providing primary-producer support for the lipid-peroxidation to OXPHOS-coupling link (Xie et al., 2018; Xie et al., 2019).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Direct studies measuring lipid peroxidation and OXPHOS coupling in the same exposure series are limited; mitochondrial dysfunction can also promote lipid peroxidation, complicating directionality.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Relationship 2203: decreased coupling of OXPHOS leads to decreased ATP pool</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP 263 reports strong evidence for this KER. Experimental studies with mitochondrial toxicants and uncouplers show concordance between impaired mitochondrial function and reduced ATP production across cell types and taxa (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). Cadmium exposure in oysters reduced state 3 respiration and affected mitochondrial bioenergetics (Sokolova et al., 2005).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Compensatory glycolysis or altered metabolic demand can obscure total ATP changes.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Relationship 2204: decreased ATP pool leads to decreased cell proliferation</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP 263 provides curated evidence that ATP depletion is associated with reduced cell proliferation, with dose and incidence concordance in several systems (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). In Chlamydomonas reinhardtii, paraquat caused ATP depletion and cell injury/death or growth-related effects in multiple-endpoint assays (Nestler et al., 2012; Jamers and De Coen, 2010).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Total ATP assays may partly reflect cell number or viability; separating ATP depletion from cytotoxicity requires careful study design.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP 263 supports this KER using evidence that reduced proliferation contributes to growth inhibition across taxa (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). Algal cell density and growth rate endpoints are directly linked to cell proliferation; reduced proliferation also supports developmental growth impairment in animals.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Growth is influenced by both cell number and cell size, as well as energy allocation, development, and environmental conditions.</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<h3><span style="font-size:18px"><span style="font-family:Calibri,sans-serif"><span style="color:#4f81bd">Inconsistencies and uncertainties</span></span></span></h3>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The main uncertainty for AOP 326 is the directionality and quantitative strength of the lipid peroxidation to OXPHOS coupling relationship. Lipid peroxidation can impair mitochondrial membranes, but mitochondrial dysfunction can also enhance ROS generation and thereby increase lipid peroxidation. The linear AOP represents one biologically plausible and empirically supported direction within a broader feedback-prone network. A second uncertainty is that mild mitochondrial uncoupling may reduce ROS production and serve as an adaptive response, whereas severe or sustained uncoupling reduces ATP synthesis and supports adverse outcomes. Finally, growth is a multifactorial endpoint; reduced cell proliferation is an important contributor, but organismal growth may also be influenced by nutrient status, development, endocrine regulation, cell death, and environmental factors.</span></span></span></p>
<h3>Quantitative Consideration</h3>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Quantitative understanding varies across the AOP. The downstream AOP 263 module has the strongest quantitative foundation, whereas upstream oxidative stress and lipid peroxidation relationships are more often described qualitatively or semi-quantitatively.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">ROS measurements are reactive, transient, and assay-dependent. Quantitative relationships can be developed within a defined assay system, but generalizable prediction across taxa and stressors remains limited (Sies et al., 2017).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Dose-response relationships are reported for oxidative stress markers and lipid peroxidation in several systems, but assay differences and lipid composition strongly affect response magnitude (Ayala et al., 2014; Knauert and Knauer, 2008).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Some quantitative associations exist between lipid peroxidation and mitochondrial membrane potential or coupling efficiency, but models generalizable across taxa and membrane types are not yet established (Moore et al., 2023; Ouillon et al., 2021).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP 263 reports strong quantitative understanding, supported by bioenergetic theory and models linking mitochondrial coupling and ATP production (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">2204: decreased ATP pool to decreased cell proliferation</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Quantitative relationships are available in defined cell systems, but thresholds depend on cell type, metabolic state, and viability effects (AOP-Wiki, 2026b; OECD, 2022).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Growth models provide quantitative relationships between proliferation and tissue or organismal growth, but extrapolation across species and exposure contexts remains uncertain (Conlon and Raff, 1999; OECD, 2022).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The following benchmark-dose/point-of-departure (BMD/POD) concordance table anchors AOP 326 to quantitative cross-KE ordering, in line with Handbook section 4C. The multiomics point-of-departure (moPOD) dataset for gamma-irradiated Daphnia magna (Song et al., 2023) provides POD magnitudes for increased ROS, decreased ATP, decreased OXPHOS coupling, and cell death, demonstrating the expected upstream-to-downstream POD ordering (more sensitive PODs upstream). The moPOD is presented as POD magnitude evidence, not as a causal re-ordering of KEs. The Lemna minor EDR50 range provides a whole-pathway apical anchor in an aquatic primary producer.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">KE 1771: ATP pool, decreased</span></span></span></p>
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP 326 can support mechanistic interpretation of growth impairment caused by oxidative stressors that induce lipid peroxidation and mitochondrial bioenergetic dysfunction. The AOP is particularly relevant for hazard identification and chemical prioritization when evidence indicates increased ROS or oxidative stress together with lipid peroxidation, mitochondrial membrane potential changes, OXPHOS impairment, ATP depletion, reduced proliferation, or growth inhibition. The AOP may also support IATA development by linking upstream NAM endpoints, such as ROS assays, lipid peroxidation markers, mitochondrial membrane potential, oxygen consumption rate, ATP content, and proliferation assays, to an apical growth endpoint.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP 326 can also support chemical grouping and read-across for stressors that share evidence of oxidative lipid damage and mitochondrial bioenergetic impairment. Because oxidative stress and lipid peroxidation are not chemical-specific, this AOP should not be used as a stand-alone basis for regulatory decisions. Instead, it should be used as part of a weight-of-evidence framework that considers stressor mode of action, exposure context, taxonomic relevance, assay specificity, and concordance across multiple KEs. The AOP also highlights important method-development needs, particularly standardized assays for lipid peroxidation, mitochondrial coupling, and ATP depletion that can be applied across taxa and integrated into quantitative AOP approaches.</span></span></span></p>
</div>
<div id="references">
<h2>References</h2>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP-Wiki. 2026a. AOP 478: Deposition of energy leading to occurrence of cataracts. Collaborative Adverse Outcome Pathway Wiki. Available from: https://aopwiki.org/aops/478.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP-Wiki. 2026b. AOP 263: Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation. Collaborative Adverse Outcome Pathway Wiki. Available from: https://aopwiki.org/aops/263.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ayala A, Munoz MF, Arguelles S. 2014. </span><span style="font-family:"Calibri",sans-serif">Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 2014:360438.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Barata C, Varo I, Navarro JC, Arun S, Porte C. 2005. Antioxidant enzyme activities and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox cycling compounds. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 140:175-186.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Bonora M, Patergnani S, Rimessi A, De Marchi E, Suski JM, Bononi A, Giorgi C, Marchi S, Missiroli S, Poletti F, Wieckowski MR, Pinton P. 2012. ATP synthesis and storage. Purinergic Signaling 8:343-357.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Carvaillo JC, Barouki R, Coumoul X, Audouze K. 2019. Linking bisphenol S to adverse outcome pathways using a combined text mining and systems biology approach. Environmental Health Perspectives 127:047005.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Cherkasov AS, Biswas PK, Ridings DM, Ringwood AH, Sokolova IM. 2006. Effects of acclimation temperature and cadmium exposure on cellular energy budgets in the marine mollusk Crassostrea virginica: linking cellular and mitochondrial responses. Journal of Experimental Biology 209:1274-1284.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Conlon I, Raff M. 1999. Size control in animal development. </span><span style="font-family:"Calibri",sans-serif">Cell 96:235-244.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Esperanza M, Cid A, Herrero C, Rioboo C. 2015. </span><span style="font-family:"Calibri",sans-serif">Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: screening cytotoxicity and genotoxicity endpoints. Aquatic Toxicology 165:210-221.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Jamers A, De Coen W. 2010. Effect assessment of the herbicide paraquat on a green alga using differential gene expression and biochemical biomarkers. Environmental Toxicology and Chemistry 29:893-901.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Jornod F, Jaylet T, Blaha L, Sarigiannis D, Tamisier L, Audouze K. 2022. AOP-helpFinder webserver: a tool for comprehensive analysis of the literature to support adverse outcome pathways development. Bioinformatics 38:1173-1175.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Knauert S, Knauer K. 2008. The role of reactive oxygen species in copper toxicity to two freshwater green microalgae. Journal of Phycology 44:311-321.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Moore TD, Martin-Creuzburg D, Yampolsky LY. 2023. Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia. Oecologia 202:151-163.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Murphy MP. 2009. How mitochondria produce reactive oxygen species. Biochemical Journal 417:1-13.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Nestler H, Groh KJ, Schonenberger R, Eggen RIL, Suter MJF. 2012. </span><span style="font-family:"Calibri",sans-serif">Multiple-endpoint assay provides a detailed mechanistic view of responses to herbicide exposure in Chlamydomonas reinhardtii. Aquatic Toxicology 110-111:214-224.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Nicotera P, Leist M, Ferrando-May E. 1998. </span><span style="font-family:"Calibri",sans-serif">Intracellular ATP, a switch in the decision between apoptosis and necrosis. Toxicology Letters 102-103:139-142. https://doi.org/10.1016/S0378-4274(98)00298-7</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">OECD. 2018. Users' handbook supplement to the guidance document for developing and assessing adverse outcome pathways. OECD Series on Adverse Outcome Pathways No. 1. Paris: OECD Publishing.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">OECD. 2021. Guidance document for the scientific review of adverse outcome pathways. OECD Series on Testing and Assessment No. 344. Paris: OECD Publishing.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">OECD. 2022. Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation. OECD Series on Adverse Outcome Pathways No. 28. Paris: OECD Publishing.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ouillon N, Sokolov EP, Otto S, Rehder G, Sokolova IM. 2021. Effects of variable oxygen regimes on mitochondrial bioenergetics and reactive oxygen species production in a marine bivalve, Mya arenaria. Journal of Experimental Biology 224:jeb237156.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Qian H, Chen W, Sun L, Jin Y, Liu W, Fu Z. 2009. Inhibitory effects of paraquat on photosynthesis and the response to oxidative stress in Chlorella vulgaris. Ecotoxicology 18:537-543.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Schieber M, Chandel NS. 2014. ROS function in redox signaling and oxidative stress. Current Biology 24:R453-R462.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Sies H, Berndt C, Jones DP. 2017. Oxidative stress. Annual Review of Biochemistry 86:715-748.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Sokolova IM, Sokolov EP, Ponnappa KM. 2005. Cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica Gmelin (Bivalvia: Ostreidae). Aquatic Toxicology 73:242-255.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Song Y, Villeneuve DL. 2021. Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation. Environmental Toxicology and Chemistry 40:2951-2963.</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Xie L, Gomes T, Solhaug KA, Song Y, Tollefsen KE. 2018. Linking mode of action of the model respiratory and photosynthesis uncoupler 3,5-dichlorophenol to adverse outcomes in Lemna minor. Aquatic Toxicology 197:98-108. https://doi.org/10.1016/j.aquatox.2018.02.005</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Xie L, Solhaug KA, Song Y, Brede DA, Lind OC, Salbu B, Tollefsen KE. 2019. Modes of action and adverse effects of gamma radiation in an aquatic macrophyte Lemna minor. Science of the Total Environment 680:23-34. https://doi.org/10.1016/j.scitotenv.2019.05.016</span></span></span></p>
<p style="margin-left:38px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Xie L, Song Y, Petersen K, Solhaug KA, Lind OC, Brede DA, Salbu B, Tollefsen KE. 2022. </span><span style="font-family:"Calibri",sans-serif">Ultraviolet B modulates gamma radiation-induced stress responses in Lemna minor at multiple levels of biological organisation. Science of the Total Environment 846:157457. https://doi.org/10.1016/j.scitotenv.2022.157457</span></span></span></p>
<td><a href="/aops/383">Aop:383 - Inhibition of Angiotensin-converting enzyme 2 leading to liver fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/382">Aop:382 - Angiotensin II type 1 receptor (AT1R) agonism leading to lung fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/384">Aop:384 - Hyperactivation of ACE/Ang-II/AT1R axis leading to chronic kidney disease </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/409">Aop:409 - Frustrated phagocytosis leads to malignant mesothelioma</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/413">Aop:413 - Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/416">Aop:416 - Aryl hydrocarbon receptor activation leading to lung cancer through IL-6 toxicity pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/418">Aop:418 - Aryl hydrocarbon receptor activation leading to impaired lung function through AHR-ARNT toxicity pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/386">Aop:386 - Deposition of ionizing energy leading to population decline via inhibition of photosynthesis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/387">Aop:387 - Deposition of ionising energy leading to population decline via mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/319">Aop:319 - Binding to ACE2 leading to lung fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/451">Aop:451 - Interaction with lung resident cell membrane components leads to lung cancer</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/476">Aop:476 - Adverse Outcome Pathways diagram related to PBDEs associated male reproductive toxicity</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/492">Aop:492 - Glutathione conjugation leading to reproductive dysfunction via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/497">Aop:497 - ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/505">Aop:505 - Reactive Oxygen Species (ROS) formation leads to cancer via inflammation pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/513">Aop:513 - Reactive Oxygen (ROS) formation leads to cancer via Peroxisome proliferation-activated receptor (PPAR) pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/521">Aop:521 - Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/462">Aop:462 - Activation of reactive oxygen species leading the atherosclerosis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/299">Aop:299 - Deposition of energy leading to population decline via DNA oxidation and follicular atresia</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/311">Aop:311 - Deposition of energy leading to population decline via DNA oxidation and oocyte apoptosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/327">Aop:327 - Excessive reactive oxygen species production leading to mortality (1)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/328">Aop:328 - Excessive reactive oxygen species production leading to mortality (2)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/329">Aop:329 - Excessive reactive oxygen species production leading to mortality (3)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/330">Aop:330 - Excessive reactive oxygen species production leading to mortality (4)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/26">Aop:26 - Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/613">Aop:613 - Peroxisome proliferator-activated receptor alpha activation leading to early life stage mortality via increased reactive oxygen species production</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/622">Aop:622 - Calcineurin inhibitor induced nephrotoxicity leading to kidney failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/636">Aop:636 - Increase in reactive oxygen species (ROS) leading to human amyotrophic lateral sclerosis (ALS)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/638">Aop:638 - Co-exposure to microplastics and cadmium leading to progression from NAFLD to liver tumorigenesis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/472">Aop:472 - DNA adduct formation leading to kidney failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell cycle disruption</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Reactive oxygen species leading to growth inhibition via protein oxidation and cell death</a></td>
<p>ROS is a normal constituent found in all organisms, <em>lifestages, and sexes.</em></p>
<h4>Key Event Description</h4>
<p>Biological State: increased reactive oxygen species (ROS)</p>
<p>Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.</p>
<p>Reactive oxygen species (ROS) are O<sub>2</sub>- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). </p>
<div>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Reactive oxygen species (ROS) refers to the chemical species superoxide, hydrogen peroxide, and their secondary reactive products. In the biological context, ROS are signaling molecules with important roles in cell energy metabolism, cell proliferation, and fate. Therefore, balancing ROS levels at the cellular and tissue level is an important part of many biological processes. Disbalance, mainly an increase in ROS levels, can cause cell dysfunction and irreversible cell damage.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are produced from both exogenous stressors and normal endogenous cellular processes, such as the mitochondrial electron transport chain (ETC). Inhibition of the ETC can result in the accumulation of ROS. Exposure to chemicals, heavy metal ions, or ionizing radiation can also result in increased production of ROS. Chemicals and heavy metal ions can deplete cellular antioxidants reducing the cell’s ability to control cellular ROS and resulting in the accumulation of ROS. Cellular antioxidants include glutathione (GSH), protein sulfhydryl groups, superoxide dismutase (SOD). </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are radicals, ions, or molecules that have a single unpaired electron in their outermost shell of electrons, which can be categorized into two groups: free oxygen radicals and non-radical ROS [Liou et al., 2010]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">highly reactive lipid- or carbohydrate-derived carbonyl compounds</span></span></p>
</td>
</tr>
</tbody>
</table>
</div>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Potential sources of ROS include NADPH oxidase, xanthine oxidase, mitochondria, nitric oxide synthase, cytochrome P450, lipoxygenase/cyclooxygenase, and monoamine oxidase [Granger et al., 2015]. ROS are generated through NADPH oxidases consisting of p47<sup>phox</sup> and p67<sup>phox</sup>. ROS are generated through xanthine oxidase activation in sepsis [Ramos et al., 2018]. Arsenic produces ROS [Zhang et al., 2011]. Mitochondria-targeted paraquat and metformin mediate ROS production [Chowdhury et al., 2020]. ROS are generated by bleomycin [Lu et al., 2010]. Radiation induces dose-dependent ROS production [Ji et al., 2019]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are generated in the course of cellular respiration, metabolism, cell signaling, and inflammation [Dickinson and Chang 2011; Egea et al. 2017]. Hydrogen peroxide is also made by the endoplasmic reticulum in the course of protein folding. Nitric oxide (NO) is produced at the highest levels by nitric oxide synthase in endothelial cells and phagocytes. NO production is one of the main mechanisms by which phagocytes kill bacteria [Wang et al., 2017]. The other species are produced by reactions with superoxide or peroxide, or by other free radicals or enzymes.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS activity is principally local. Most ROS have short half-lives, ranging from nano- to milliseconds, so diffusion is limited, while reactive nitrogen species (RNS) nitric oxide or peroxynitrite can survive long enough to diffuse across membranes [Calcerrada et al. 2011]. Consequently, local concentrations of ROS are much higher than average cellular concentrations, and signaling is typically controlled by colocalization with redox buffers [Dickinson and Chang 2011; Egea et al. 2017]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Although their existence is limited temporally and spatially, ROS interact with other ROS or with other nearby molecules to produce more ROS and participate in a feedback loop to amplify the ROS signal, which can increase RNS. Both ROS and RNS also move into neighboring cells, and ROS can increase intracellular ROS signaling in neighboring cells [Egea et al. 2017].</span></span></p>
<p>In the primary event, photoreactive chemicals are excited by the absorption of photon energy. The energy of the photoactivated chemicals transfer to oxygen and then generates the reactive oxygen species (ROS), including superoxide (O<sub>2</sub><sup>−</sup>) via type I reaction and singlet oxygen (<sup>1</sup>O<sub>2</sub>) via type II reaction, as principal intermediate species in phototoxic reaction (Foote, 1991, Onoue et al. , 2009).</p>
</div>
<h4>How it is Measured or Detected</h4>
<p>Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies.</p>
<p>Yuan, Yan, et al., (2013) described ROS monitoring by using H<sub>2</sub>-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H<sub>2</sub>-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm.</p>
<p>Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013).</p>
<p>Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS.</p>
<p>On the basis of the pathogenesis of drug-induced phototoxicity, a reactive oxygen species (ROS) assay was proposed to evaluate the phototoxic risk of chemicals. The ROS assay can monitor generation of ROS, such as singlet oxygen and superoxide, from photoirradiated chemicals, and the ROS data can be used to evaluate the photoreactivity of chemicals (Onoue et al. , 2014, Onoue et al. , 2013, Onoue and Tsuda, 2006). The ROS assay is a recommended approach by guidelines to evaluate the phototoxic risk of chemicals (ICH, 2014, PCPC, 2014).</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Many fluorescent compounds can be used to detect ROS, some of which are specific, and others are less specific. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・ROS can be detected by fluorescent probes such as <em>p</em>-methoxy-phenol derivative [Ashoka et al., 2020].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Chemiluminescence analysis can detect the superoxide, where some probes have a wider range for detecting hydroxyl radical, hydrogen peroxide, and peroxynitrite [Fuloria et al., 2021].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・ROS in the blood can be detected using superparamagnetic iron oxide nanoparticles (SPION)-based biosensor [Lee et al., 2020].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) can be detected with a colorimetric probe, which reacts with H<sub>2</sub>O<sub>2</sub> in a 1:1 stoichiometry to produce a bright pink colored product, followed by the detection with a standard colorimetric microplate reader with a filter in the 540-570 nm range.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・The levels of ROS can be quantified using multiple-step amperometry using a stainless steel counter electrode and non-leak Ag|AgCl reference node [Flaherty et al., 2017].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Singlet oxygen can be measured by monitoring the bleaching of <em>p</em>-nitrosodimethylaniline at 440 nm using a spectrophotometer with imidazole as a selective acceptor of singlet oxygen [Onoue et al., 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Alternative methods involve the detection of redox-dependent changes to cellular constituents such as proteins, DNA, lipids, or glutathione [Dickinson and Chang 2011; Wang et al. 2013; Griendling et al. 2016]. However, these methods cannot generally distinguish between the oxidative species behind the changes and cannot provide good resolution for the kinetics of oxidative activity.</span></span></p>
</div>
<h4>References</h4>
<p>Akai, K., et al. (2004). "Ability of ferric nitrilotriacetate complex with three pH-dependent conformations to induce lipid peroxidation." Free Radic Res. Sep;38(9):951-62. doi: 10.1080/1071576042000261945</p>
<p>Ashoka, A. H., et al. (2020). "Recent Advances in Fluorescent Probes for Detection of HOCl and HNO." ACS omega, 5(4), 1730-1742. doi:10.1021/acsomega.9b03420</p>
<p>B.H. Park, S.M. Fikrig, E.M. Smithwick Infection and nitroblue tetrazolium reduction by neutrophils: a diagnostic aid Lancet, 2 (1968), pp. 532-534</p>
<p>Bedard, Karen, and Karl-Heinz Krause. 2007. “The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology.” Physiological Reviews 87 (1): 245–313.</p>
<p>Bisht, Shilpa, Muneeb Faiq, Madhuri Tolahunase, and Rima Dada. 2017. “Oxidative Stress and Male Infertility.” Nature Reviews. Urology 14 (8): 470–85.</p>
<p>Brieger, K., S. Schiavone, F. J. Miller Jr, and K-H Krause. 2012. “Reactive Oxygen Species: From Health to Disease.” Swiss Medical Weekly 142 (August): w13659.</p>
<p>Calcerrada, P., et al. (2011). "Nitric oxide-derived oxidants with a focus on peroxynitrite: molecular targets, cellular responses and therapeutic implications." Curr Pharm Des 17(35): 3905-3932.</p>
<p>Chattopadhyay, Sukumar, et al. "Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants." Toxicology letters 136.1 (2002): 65-76.</p>
<p>Chowdhury, A. R., et al. (2020). "Mitochondria-targeted paraquat and metformin mediate ROS production to induce multiple pathways of retrograde signaling: A dose-dependent phenomenon." Redox Biol. doi: 10.1016/j.redox.2020.101606. PMID: 32604037; PMCID: PMC7327929.</p>
<p>Dickinson, B. C. and Chang C. J. (2011). "Chemistry and biology of reactive oxygen species in signaling or stress responses." Nature chemical biology 7(8): 504-511.</p>
<p>Drew, Barry, and Christiaan Leeuwenburgh. 2002. “Aging and the Role of Reactive Nitrogen Species.” Annals of the New York Academy of Sciences 959 (April): 66–81.</p>
<p>Egea, J., et al. (2017). "European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)." Redox biology 13: 94-162.</p>
<p>Flaherty, R. L., et al. (2017). "Glucocorticoids induce production of reactive oxygen species/reactive nitrogen species and DNA damage through an iNOS mediated pathway in breast cancer." Breast Cancer Research, 19(1), 1–13. https://doi.org/10.1186/s13058-017-0823-8</p>
<p>Foote CS. Definition of type I and type II photosensitized oxidation. Photochem Photobiol. 1991;54:659.</p>
<p>Fuloria, S., et al. (2021). "Comprehensive Review of Methodology to Detect Reactive Oxygen Species (ROS) in Mammalian Species and Establish Its Relationship with Antioxidants and Cancer." Antioxidants (Basel, Switzerland) 10(1) 128. doi:10.3390/antiox10010128</p>
<p>Go, Y. M. and Jones, D. P. (2013). "The redox proteome." J Biol Chem 288(37): 26512-26520.</p>
<p>Goud, Anuradha P., Pravin T. Goud, Michael P. Diamond, Bernard Gonik, and Husam M. Abu-Soud. 2008. “Reactive Oxygen Species and Oocyte Aging: Role of Superoxide, Hydrogen Peroxide, and Hypochlorous Acid.” Free Radical Biology & Medicine 44 (7): 1295–1304.</p>
<p>Granger, D. N. and Kvietys, P. R. (2015). "Reperfusion injury and reactive oxygen species: The evolution of a concept" Redox Biol. doi: 10.1016/j.redox.2015.08.020. PMID: 26484802; PMCID: PMC4625011.</p>
<p>Griendling, K. K., et al. (2016). "Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association." Circulation research 119(5): e39-75.</p>
<p>Griendling, Kathy K., Rhian M. Touyz, Jay L. Zweier, Sergey Dikalov, William Chilian, Yeong-Renn Chen, David G. Harrison, Aruni Bhatnagar, and American Heart Association Council on Basic Cardiovascular Sciences. 2016. “Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association.” Circulation Research 119 (5): e39–75.</p>
<p>ICH. ICH Guideline S10 Guidance on Photosafety Evaluation of Pharmaceuticals.: International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; 2014.</p>
<p>Itziou, A., et al. (2011). "In vivo and in vitro effects of metals in reactive oxygen species production, protein carbonylation, and DNA damage in land snails Eobania vermiculata." Archives of Environmental Contamination and Toxicology, 60(4), 697–707. https://doi.org/10.1007/s00244-010-9583-5</p>
<p>Ji, W. O., et al. "Quantitation of the ROS production in plasma and radiation treatments of biotargets." Sci Rep. 2019 Dec 27;9(1):19837. doi: 10.1038/s41598-019-56160-0. PMID: 31882663; PMCID: PMC6934759.</p>
<p>Kruk, J. and Aboul-Enein, H. Y. (2017). "Reactive Oxygen and Nitrogen Species in Carcinogenesis: Implications of Oxidative Stress on the Progression and Development of Several Cancer Types." Mini-Reviews in Medicinal Chemistry, 17:11. doi:10.2174/1389557517666170228115324</p>
<p>Lee, D. Y., et al. (2020). "PEGylated Bilirubin-coated Iron Oxide Nanoparticles as a Biosensor for Magnetic Relaxation Switching-based ROS Detection in Whole Blood." Theranostics, 10(5), 1997-2007. doi:10.7150/thno.39662</p>
<p>Li, Z., et al. (2020). "Inhibition of MiR-25 attenuates doxorubicin-induced apoptosis, reactive oxygen species production and DNA damage by targeting pten." International Journal of Medical Sciences, 17(10), 1415–1427. https://doi.org/10.7150/ijms.41980</p>
<p>Liou, G. Y. and Storz, P. "Reactive oxygen species in cancer." Free Radic Res. 2010 May;44(5):479-96. doi:10.3109/10715761003667554. PMID: 20370557; PMCID: PMC3880197.</p>
<p>Lu, Y., et al. (2010). "Phosphatidylinositol-3-kinase/akt regulates bleomycin-induced fibroblast proliferation and collagen production." American journal of respiratory cell and molecular biology, 42(4), 432–441. https://doi.org/10.1165/rcmb.2009-0002OC</p>
<p>Onoue, S., et al. (2013). "Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation." J Appl Toxicol. 33(11):1241-50. doi: 10.1002/jat.2776. Epub 2012 Jun 13. PMID: 22696462.</p>
<p>Onoue S, Hosoi K, Toda T, Takagi H, Osaki N, Matsumoto Y, et al. Intra-/inter-laboratory validation study on reactive oxygen species assay for chemical photosafety evaluation using two different solar simulators. Toxicology in vitro : an international journal published in association with BIBRA. 2014;28:515-23.</p>
<p>Onoue S, Hosoi K, Wakuri S, Iwase Y, Yamamoto T, Matsuoka N, et al. Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation. Journal of applied toxicology : JAT. 2013;33:1241-50.</p>
<p>Onoue S, Kawamura K, Igarashi N, Zhou Y, Fujikawa M, Yamada H, et al. Reactive oxygen species assay-based risk assessment of drug-induced phototoxicity: classification criteria and application to drug candidates. J Pharm Biomed Anal. 2008;47:967-72.</p>
<p>Onoue S, Seto Y, Gandy G, Yamada S. Drug-induced phototoxicity; an early<em> in vitro</em> identification of phototoxic potential of new drug entities in drug discovery and development. Current drug safety. 2009;4:123-36.</p>
<p>Onoue S, Tsuda Y. Analytical studies on the prediction of photosensitive/phototoxic potential of pharmaceutical substances. Pharmaceutical research. 2006;23:156-64.</p>
<p>Ozcan, Ayla, and Metin Ogun. 2015. “Biochemistry of Reactive Oxygen and Nitrogen Species.” In Basic Principles and Clinical Significance of Oxidative Stress, edited by Sivakumar Joghi Thatha Gowder. Rijeka: IntechOpen.</p>
<p>Parrish, A. R. 2010. “2.27 - Hypoxia/Ischemia Signaling.” In Comprehensive Toxicology (Second Edition), edited by Charlene A. McQueen, 529–42. Oxford: Elsevier.</p>
<p>PCPC. PCPC 2014 safety evaluation guidelines; Chapter 7: Evaluation of Photoirritation and Photoallergy potential. Personal Care Products Council; 2014.</p>
<p>Ramos, M. F. P., et al. (2018). "Xanthine oxidase inhibitors and sepsis." Int J Immunopathol Pharmacol. 32:2058738418772210. doi:10.1177/2058738418772210</p>
<p>Ravanat, J. L., et al. (2014). "Radiation-mediated formation of complex damage to DNA: a chemical aspect overview." Br J Radiol 87(1035): 20130715.</p>
<p>Schutzendubel, A. and Polle, A. (2002). "Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization." Journal of Experimental Botany, 53(372), 1351–1365. https://doi.org/10.1093/jexbot/53.372.1351</p>
<p>Seto Y, Kato M, Yamada S, Onoue S. Development of micellar reactive oxygen species assay for photosafety evaluation of poorly water-soluble chemicals. Toxicology in vitro : an international journal published in association with BIBRA. 2013;27:1838-46.</p>
<p>Sharma, Gunjan, Nishant Kumar Rana, Priya Singh, Pradeep Dubey, Daya Shankar Pandey, and Biplob Koch. 2017. “p53 Dependent Apoptosis and Cell Cycle Delay Induced by Heteroleptic Complexes in Human Cervical Cancer Cells.” Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie 88 (April): 218–31.</p>
<p>Silva, R., et al. (2019). "Light exposure during growth increases riboflavin production, reactive oxygen species accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains." FEMS Yeast Research, 19(1), 1–7. https://doi.org/10.1093/femsyr/foy114</p>
<p>Tsuchiya K, et al. (2005). "Oxygen radicals photo-induced by ferric nitrilotriacetate complex." Biochim Biophys Acta. 1725(1):111-9. doi:10.1016/j.bbagen.2005.05.001</p>
<p>Wang, J., et al. (2017). "Glucocorticoids Suppress Antimicrobial Autophagy and Nitric Oxide Production and Facilitate Mycobacterial Survival in Macrophages." Scientific reports, 7(1), 982. https://doi.org/10.1038/s41598-017-01174-9</p>
<p>Wang, X., et al. (2013). "Imaging ROS signaling in cells and animals." Journal of molecular medicine 91(8): 917-927.</p>
<p>Yen, Cheng Chien, et al. "Inorganic arsenic causes cell apoptosis in mouse cerebrum through an oxidative stress-regulated signaling pathway." Archives of toxicology 85 (2011): 565-575.</p>
<p>Yuan, Yan, et al. "Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway." PloS one 8.5 (2013): e64330.</p>
<p>Zhang, Z., et al. (2011). "Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/β-catenin pathway in human colorectal adenocarcinoma DLD1 cells. " Toxicology and Applied Pharmacology, 256(2), 114-121. doi:10.1016/j.taap.2011.07.016</p>
<td><a href="/aops/220">Aop:220 - Cyp2E1 Activation Leading to Liver Cancer</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/17">Aop:17 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress during brain development leads to impairment of learning and memory</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/284">Aop:284 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress leads to chronic kidney disease</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/411">Aop:411 - Oxidative stress Leading to Decreased Lung Function </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/424">Aop:424 - Oxidative stress Leading to Decreased Lung Function via CFTR dysfunction</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/425">Aop:425 - Oxidative Stress Leading to Decreased Lung Function via Decreased FOXJ1</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/429">Aop:429 - A cholesterol/glucose dysmetabolism initiated Tau-driven AOP toward memory loss (AO) in sporadic Alzheimer's Disease with plausible MIE's plug-ins for environmental neurotoxicants</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/452">Aop:452 - Adverse outcome pathway of PM-induced respiratory toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/464">Aop:464 - Calcium overload in dopaminergic neurons of the substantia nigra leading to parkinsonian motor deficits</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/470">Aop:470 - Deposition of energy leads to abnormal vascular remodeling</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/478">Aop:478 - Deposition of energy leading to occurrence of cataracts</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/479">Aop:479 - Mitochondrial complexes inhibition leading to left ventricular function decrease via increased myocardial oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/481">Aop:481 - AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/482">Aop:482 - Deposition of energy leading to occurrence of bone loss</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/483">Aop:483 - Deposition of Energy Leading to Learning and Memory Impairment</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/505">Aop:505 - Reactive Oxygen Species (ROS) formation leads to cancer via inflammation pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/521">Aop:521 - Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/26">Aop:26 - Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/488">Aop:488 - Increased reactive oxygen species production leading to decreased cognitive function</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/437">Aop:437 - Inhibition of mitochondrial electron transport chain (ETC) complexes leading to kidney toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/535">Aop:535 - Binding and activation of GPER leading to learning and memory impairments</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/171">Aop:171 - Chronic cytotoxicity of the serous membrane leading to pleural/peritoneal mesotheliomas in the rat.</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/138">Aop:138 - Organic anion transporter (OAT1) inhibition leading to renal failure and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/177">Aop:177 - Cyclooxygenase 1 (COX1) inhibition leading to renal failure and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/186">Aop:186 - unknown MIE leading to renal failure and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/200">Aop:200 - Estrogen receptor activation leading to breast cancer </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/444">Aop:444 - Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/447">Aop:447 - Kidney failure induced by inhibition of mitochondrial electron transfer chain through apoptosis, inflammation and oxidative stress pathways</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/476">Aop:476 - Adverse Outcome Pathways diagram related to PBDEs associated male reproductive toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/497">Aop:497 - ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/457">Aop:457 - Succinate dehydrogenase inhibition leading to increased insulin resistance through reduction in circulating thyroxine</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/459">Aop:459 - AhR activation in the thyroid leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/507">Aop:507 - Nrf2 inhibition leading to vascular disrupting effects via inflammation pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/509">Aop:509 - Nrf2 inhibition leading to vascular disrupting effects through activating apoptosis signal pathway and mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/510">Aop:510 - Demethylation of PPAR promotor leading to vascular disrupting effects</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/511">Aop:511 - The AOP framework on ROS-mediated oxidative stress induced vascular disrupting effects </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/538">Aop:538 - Adverse outcome pathway of PFAS-induced vascular disrupting effects via activating oxidative stress related pathways </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/260">Aop:260 - CYP2E1 activation and formation of protein adducts leading to neurodegeneration</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/450">Aop:450 - Inhibition of AChE and activation of CYP2E1 leading to sensory axonal peripheral neuropathy and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/501">Aop:501 - Excessive iron accumulation leading to neurological disorders</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/471">Aop:471 - Neuron defect induced early behavioral change</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/31">Aop:31 - Oxidation of iron in hemoglobin leading to hematotoxicity</a></td>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/616">Aop:616 - organic UV filter and its Photoproducts reproductive toxicity pathways </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/622">Aop:622 - Calcineurin inhibitor induced nephrotoxicity leading to kidney failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/625">Aop:625 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/628">Aop:628 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/472">Aop:472 - DNA adduct formation leading to kidney failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/642">Aop:642 - Intestinal FXR inhibition leading to steatohepatitis via gut‑liver axis dysregulation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell cycle disruption</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Reactive oxygen species leading to growth inhibition via protein oxidation and cell death</a></td>
<p><span style="color:#27ae60"><strong>Taxonomic applicability: </strong>Occurrence of oxidative stress is not species specific. </span></p>
<p><span style="color:#27ae60"><strong>Life stage applicability:</strong> Occurrence of oxidative stress is not life stage specific. </span></p>
<p><span style="color:#27ae60"><strong>Sex applicability: </strong>Occurrence of oxidative stress is not sex specific. </span></p>
<p><span style="color:#27ae60"><strong>Evidence for perturbation by prototypic stressor:</strong> There is evidence of the increase of oxidative stress following perturbation from a variety of stressors including exposure to ionizing radiation and altered gravity (Bai et al., 2020; Ungvari et al., 2013; Zhang et al., 2009). </span></p>
<h4>Key Event Description</h4>
<p>Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell. As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al., 2009) and can be used as indicators of the presence of oxidative stress in the cell. </p>
<p>In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides on neighboring amino acids (Antelmann & Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010). </p>
<p>ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol, or antioxidant vitamins and minerals including vitamin E, C, carotene, lutein, zeaxanthin, selenium, and zinc (Fletcher, 2010). The enzymes, vitamins and minerals catalyze the conversion of ROS to non-toxic molecules such as water and O2. However, these antioxidant systems are not perfect and endogenous metabolic processes and/or exogenous oxidative influences can trigger cumulative oxidative injuries to the mitochondria, causing a decline in their functionality and efficiency, which further promotes cellular oxidative stress (Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017). </p>
<p>However, an emerging viewpoint suggests that ROS-induced modifications may not be as detrimental as previously thought, but rather contribute to signaling processes (Foyer et al., 2017). </p>
<p> </p>
<p><strong>Sources of ROS Production </strong></p>
<p><strong>Direct Sources: </strong>Direct sources involve the deposition of energy onto water molecules, breaking them into active radical species. When ionizing radiation hits water, it breaks it into hydrogen (H*) and hydroxyl (OH*) radicals by destroying its bonds. The hydrogen will create hydroxyperoxyl free radicals (HO2*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H2O2) and more O2 (Elgazzar and Kazem, 2015). Antioxidant mechanisms are also affected by radiation, with catalase (CAT) and peroxidase (POD) levels rising as a result of exposure (Seen et al. 2018; Ahmad et al. 2021). </p>
<p><strong>Indirect Sources</strong>: An indirect source of ROS is the mitochondria, which is one of the primary producers in eukaryotic cells (Powers et al., 2008). As much as 2% of the electrons that should be going through the electron transport chain in the mitochondria escape, allowing them an opportunity to interact with surrounding structures. Electron-oxygen reactions result in free radical production, including the formation of hydrogen peroxide (H2O2) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O2, and inorganic phosphate (Pi) (Hargreaves et al. 2020; Raimondi et al. 2020; Vargas-Mendoza et al. 2021). The first and third complexes of the transport chain are the most relevant to mammalian ROS production (Raimondi et al., 2020). The mitochondria has its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS is also produced through nicotinamide adenine dinucleotide phosphate oxidase (Nox) stimulation, an event commenced by angiotensin II, a product/effector of the renin-angiotensin system (Nguyen Dinh Cat et al. 2013; Forrester et al. 2018). Other ROS producers include xanthine oxidase, immune cells (macrophage, neutrophils, monocytes, and eosinophils), phospholipase A2 (PLA2), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021). </p>
<h4>How it is Measured or Detected</h4>
<p><strong>Oxidative Stress:</strong> Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed </p>
<ul>
<li>Detection of ROS by chemiluminescence (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>
<p> </p>
<p><strong>Molecular Biology:</strong> Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include: </p>
<ul>
<li>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus 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 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>
</ul>
<p>In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation.</p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong>Assay Type & Measured Content </strong></p>
</td>
<td>
<p><strong>Description </strong></p>
</td>
<td>
<p><strong>Dose Range Studied </strong></p>
</td>
<td>
<p><strong>Assay Characteristics (Length/Ease of use/Accuracy) </strong></p>
</td>
</tr>
<tr>
<td>
<p>ROS </p>
<p>Formation in the Mitochondria assay (Shaki et al., 2012) </p>
</td>
<td>
<p>“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 µM) in respiration buffer containing (0.32 mM sucrose, 10mM Tris, 20 mM Mops, 50 µM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 µM) to mitochondria and was then incubated for 10 min.Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.” </p>
<p> </p>
</td>
<td>
<p>0, 50,100 and 200 µM of Uranyl Acetate </p>
<p> </p>
</td>
<td>
<p> Long/ Easy High accuracy </p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Mitochondrial Antioxidant Content Assay Measuring GSH content (Shaki et al., 2012) </p>
<p> </p>
</td>
<td>
<p>“GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as µg/mg protein.” </p>
</td>
<td>
<p>0, 50, </p>
<p>100, or </p>
<p>200 µM </p>
<p>Uranyl Acetate </p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>H2O2 Production Assay Measuring H2O2 Production in isolated mitochondria (Heyno et al., 2008) </p>
<p> </p>
</td>
<td>
<p>“Effect of CdCl2 and antimycin A (AA) on H2O2 production in isolated mitochondria from potato. H2O2 production was measured as scopoletin oxidation. Mitochondria were incubated for 30 min in the measuring buffer </p>
<p>(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.” </p>
</td>
<td>
<p>0, 10, 30 </p>
<p>µM Cd2+ </p>
<p> </p>
<p>2 µM antimycin A </p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Flow Cytometry ROS & Cell Viability (Kruiderig et al., 1997) </p>
<p> </p>
</td>
<td>
<p>“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)” </p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>Strong/easy medium </p>
</td>
</tr>
<tr>
<td>
<p>DCFH-DA </p>
<p>Assay Detection of hydrogen peroxide production (Yuan et al., </p>
<p>2016) </p>
</td>
<td>
<p>Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H2O2 to form fluorescent production. </p>
<p> </p>
</td>
<td>
<p>0-400 </p>
<p>µM </p>
</td>
<td>
<p>Long/ Easy High accuracy </p>
</td>
</tr>
<tr>
<td>
<p>H2-DCF-DAAssay Detection of superoxide production (Thiebault etal., 2007) </p>
<p> </p>
</td>
<td>
<p>This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer. </p>
<p>The dye (CM-H2DCFDA) diffuses into the cell and is cleaved by esterases, the thiol reactive chlormethyl group reacts with intracellular glutathione which can be detected using flow cytometry. </p>
</td>
<td>
<p> </p>
</td>
<td>
<p>Long/Easy/ High Accuracy </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong>Method of Measurement </strong></p>
</td>
<td>
<p><strong>References </strong></p>
</td>
<td>
<p><strong>Description </strong></p>
</td>
<td colspan="2">
<p><strong>OECD-Approved Assay </strong></p>
</td>
</tr>
<tr>
<td>
<p>Chemiluminescence </p>
</td>
<td>
<p>(Lu, C. et al., 2006; </p>
<p>Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>ROS can induce electron transitions in molecules, leading to electronically excited products. When the electrons transition back to ground state, chemiluminescence is emitted and can be measured. Reagents such as luminol and lucigenin are commonly used to amplify the signal. </p>
</td>
<td colspan="2">
<p>No </p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Spectrophotometry </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>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. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Direct or Spin Trapping-Based electron paramagnetic resonance (EPR) Spectroscopy </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>The unpaired electrons (free radicals) found in ROS can be detected with EPR and is known as electron paramagnetic resonance. A variety of spin traps can be used. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Nitroblue Tetrazolium Assay </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>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. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>Fluorescence analysis of DHE is used to measure O2.− levels. O2.− is reduced to O2 as DHE is oxidized to 2-hydroxyethidium, and this reaction can be measured by fluorescence. Similarly, hydrocyans can be oxidized by any ROS, and measured via fluorescence. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Amplex Red Assay </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>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. </p>
<p>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. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>HyPer Probe </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>Fluorescent measurement of intracellular H2O2 levels. HyPer is a genetically encoded fluorescent sensor that can be used for in vivo and in situ imaging. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Cytochrome c Reduction Assay </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>The cytochrome c reduction assay is used to measure O2.− levels. O 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. </p>
<p>The redox state of tissue is detected through nuclear magnetic resonance/magnetic resonance imaging, with the use of a nitroxide spin probe or biradical molecule. </p>
</td>
<td colspan="2">
<p>No </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Glutathione (GSH) depletion </p>
</td>
<td>
<p>(Biesemann, N. et al., 2018) </p>
</td>
<td>
<p>A downstream target of the Nrf2 pathway is involved in GSH synthesis. As an indication of oxidation status, GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., <a href="http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html" rel="noreferrer noopener" target="_blank">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</a>). </p>
<p>Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Protein oxidation (carbonylation) </p>
</td>
<td>
<p>(Azimzadeh et al., 2017; Azimzadeh et al., 2015; Ping et al., 2020) </p>
</td>
<td>
<p>Can be determined with ELISA or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Seahorse XFp Analyzer </p>
</td>
<td>
<p>Leung et al. 2018 </p>
</td>
<td>
<p>The Seahorse XFp Analyzer provides information on mitochondrial function, oxidative stress, and metabolic dysfunction of viable cells by measuring respiration (oxygen consumption rate; OCR) and extracellular pH (extracellular acidification rate; ECAR). </p>
</td>
<td>
<p>No </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include: </p>
<table border="1">
<tbody>
<tr>
<td>
<p>Method of Measurement </p>
</td>
<td>
<p>References </p>
</td>
<td>
<p>Description </p>
</td>
<td>
<p>OECD-Approved Assay </p>
</td>
</tr>
<tr>
<td>
<p>Immunohistochemistry </p>
</td>
<td>
<p>(Amsen, D., de Visser, K. E., and Town, T., 2009) </p>
</td>
<td>
<p>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>qPCR </p>
</td>
<td>
<p>(Forlenza et al., 2012) </p>
</td>
<td>
<p>qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences) </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis </p>
</td>
<td>
<p>(Jackson, A. F. et al., 2014) </p>
</td>
<td>
<p>Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway </p>
</td>
<td>
<p>No </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<h4>References</h4>
<p>Ahmad, S. et al. (2021), “60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression”, Journal Insect Science, Vol. 21/5, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jisesa/ieab080" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jisesa/ieab080</a> </p>
<p>Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, Antioxidants & Redox Signaling, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3400" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2010.3400</a> </p>
<p>Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, <a href="https://doi.org/10.1007/978-1-59745-447-6_5" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/978-1-59745-447-6_5</a> </p>
<p>Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, <a href="https://doi.org/10.1021/pr501141b" rel="noreferrer noopener" target="_blank">https://doi.org/10.1021/pr501141b</a> </p>
<p>Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, <a href="https://doi.org/10.1080/09553002.2017.1339332" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2017.1339332</a> </p>
<p>Azzam, E. I. et al. (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, Cancer Letters, Vol. 327/1-2, Elsevier, Ireland, https://doi.org/10.1016/j.canlet.2011.12.012 </p>
<p>Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, American Journal of Physiology - Cell Physiology, Vol. 318/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/ajpcell.00520.2019." rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/ajpcell.00520.2019.</a> </p>
<p>Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, Journal of ocular pharmacology and therapeutics, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, <a href="https://doi.org/10.1089/jop.2000.16.285.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a> </p>
<p>Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”, Scientific Reports, Vol. 8/1, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41598-018-27614-8" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41598-018-27614-8</a>. </p>
<p>Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in The Pathophysiologic Basis of Nuclear Medicine, Springer, New York, pp. 540-548 </p>
<p>Eruslanov, E., & Kusmartsev, S. (2010). Identification of ROS using oxidized DCFDA and flow-cytometry. Methods in molecular biology ,N.J., Vol. 594, https://doi.org/10.1007/978-1-60761-411-1_4 </p>
<p>Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, Ophthalmic Research, Vol. 44, Karger International, Basel, <a href="https://doi.org/10.1159/000316476.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a> </p>
<p>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, https://doi.org/10.1007/978-1-61779-439-1_2 </p>
<p>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" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/physrev.00038.201</a> </p>
<p>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 </p>
<p>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> </p>
<p>Griendling, K. K. et al. (2016), “Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association”, Circulation research, Vol. 119/5, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/RES.0000000000000110" rel="noreferrer noopener" target="_blank">https://doi.org/10.1161/RES.0000000000000110</a> </p>
<p>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" rel="noreferrer noopener" target="_blank">https://doi.org/10.3969/j.issn.1673-5374.2013.21.009</a> </p>
<p>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" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s42255-020-0251-4</a> </p>
<p>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" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.mrrev.2016.08.003</a> </p>
<p>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" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2010.3222</a> </p>
<p>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" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.taap.2013.10.019</a> </p>
<p>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" rel="noreferrer noopener" target="_blank">https://doi.org/10.1002/em.20406</a> </p>
<p>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> </p>
<p>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 </p>
<p>Lu, C., G. Song, and J. Lin (2006), “Reactive oxygen species and their chemiluminescence-detection methods”, TrAC Trends in Analytical Chemistry, Vol. 25/10, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.trac.2006.07.007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.trac.2006.07.007</a> </p>
<p>Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH oxidase, and redox signaling in the vasculature”, Antioxidants & redox signaling, Vol. 19/10, Mary Ann Liebert, Larchmont, <a href="https://doi.org/10.1089/ars.2012.4641" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2012.4641</a> </p>
<p>Ping, Z. et al. (2020), “Oxidative Stress in Radiation-Induced Cardiotoxicity”, Oxidative Medicine and Cellular Longevity, Vol. 2020, Hindawi, <a href="https://doi.org/10.1155/2020/3579143" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2020/3579143</a> </p>
<p>Powers, S.K. and M.J. Jackson. (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, Physiological Reviews, Vol. 88/4, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00031.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/physrev.00031.2007</a> </p>
<p>Raimondi, V., F. Ciccarese and V. Ciminale. (2020), “Oncogenic pathways and the electron transport chain: a dangeROS liason”, British Journal of Cancer, Vol. 122/2, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41416-019-0651-y" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41416-019-0651-y</a> </p>
<p>Seen, S. and L. Tong. (2018), “Dry eye disease and oxidative stress”, Acta Ophthalmologica, Vol. 96/4, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1111/aos.13526" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/aos.13526</a> </p>
<p>Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, Vol. 68/12, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/gerona/glt057." rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/gerona/glt057.</a> </p>
<p> </p>
<p>Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, Life, Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/life11111269" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/life11111269</a> </p>
<p>Wang, H. et al. (2019), “Radiation-induced heart disease: a review of classification, mechanism and prevention”, International Journal of Biological Sciences, Vol. 15/10, Ivyspring International Publisher, Sydney, <a href="https://doi.org/10.7150/ijbs.35460" rel="noreferrer noopener" target="_blank">https://doi.org/10.7150/ijbs.35460</a> </p>
<p>Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, Journal of applied physiology, Vol. 106/1, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.01278.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/japplphysiol.01278.2007</a>. </p>
<p>Zhao, R. Z. et al. (2019), “Mitochondrial electron transport chain, ROS generation and uncoupling”, International journal of molecular medicine, Vol. 44/1, Spandidos Publishing Ltd., Athens, <a href="https://doi.org/10.3892/ijmm.2019.4188" rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/ijmm.2019.4188</a> </p>
<td><a href="/aops/329">Aop:329 - Excessive reactive oxygen species production leading to mortality (3)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/413">Aop:413 - Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/492">Aop:492 - Glutathione conjugation leading to reproductive dysfunction via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/521">Aop:521 - Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/615">Aop:615 - Suppression of Keap1 cysteine oxidation leading to liver inflammation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The biological domain of applicability for this KE is broad because lipid membranes and oxidizable fatty acids are widely conserved biological features. The event is applicable wherever lipid substrates susceptible to oxidation are present and where oxidants can access those substrates. The KE is therefore relevant across many biological systems, including unicellular algae, invertebrates, fish, mammals and human-derived cells. The current evidence base is strongest in mammalian systems because lipid peroxidation chemistry and analytical methods have been extensively studied there, but ecotoxicological evidence supports relevance in algae, crustaceans, mollusks and fish.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">The KE is not intrinsically limited by sex or life stage. However, the magnitude of lipid peroxidation and its downstream consequences may be modified by lipid composition, antioxidant capacity, oxygen availability, temperature, metabolic rate, nutritional status, metal availability, and exposure duration. Organisms or tissues enriched in polyunsaturated fatty acids, exposed to high oxygen flux, or experiencing antioxidant depletion may be particularly susceptible. In photosynthetic organisms, lipid peroxidation may also occur in chloroplast and thylakoid membranes; in animals, mitochondria and plasma membranes are common sites of interest.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">Within the ROS-growth AOP network, this KE is especially relevant as a molecular damage event linking oxidative stress to impaired mitochondrial membrane function, decreased coupling of oxidative phosphorylation, reduced ATP production, cell injury, and decreased growth. Nevertheless, this KE should remain modular: it may be reused in other AOPs whenever increased lipid oxidation products are measured as a consequence of oxidative stress or other lipid-damaging perturbations.</span></span></span></p>
<p style="text-align:justify"> </p>
<h4>Key Event Description</h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Lipid peroxidation is an oxidative degradation process affecting lipids, particularly polyunsaturated fatty acids in cellular and organelle membranes. The process is initiated when oxidants, including free radicals and reactive oxygen species, abstract hydrogen atoms from susceptible lipid chains. This generates lipid radicals that react with molecular oxygen to form lipid peroxyl radicals and lipid hydroperoxides. These products can propagate chain reactions, producing additional oxidized lipids and secondary reactive aldehydes such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), and related hydroxyalkenals (Esterbauer et al., 1991; Yin et al., 2011; Ayala et al., 2014).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">As a key event, increased lipid peroxidation represents a measurable increase in oxidized lipid products relative to an appropriate control state. The event may reflect direct oxidative damage to membrane lipids, increased formation of lipid hydroperoxides, increased accumulation of MDA or 4-HNE, or increased abundance of specific oxidized phospholipid or fatty acid species. Because lipid peroxidation products can alter membrane fluidity, permeability and signaling, the event is relevant both as a marker of oxidative damage and as a potential contributor to downstream mitochondrial dysfunction, loss of membrane integrity, cytotoxicity and impaired growth (Esterbauer et al., 1991; Uchida, 2003; Ayala et al., 2014).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:"Calibri",sans-serif">This KE should be described independently of any specific upstream or downstream event. In an AOP context, lipid peroxidation is commonly downstream of oxidative stress and upstream of events related to decreased mitochondrial coupling, cellular injury, or altered membrane-dependent biological processes. However, the KE itself is defined only by the increased lipid oxidation state and its measurable biochemical products.</span></span></p>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><strong><span style="font-family:"Calibri",sans-serif">No OECD Test Guideline is currently dedicated specifically to measurement of lipid peroxidation as a standalone endpoint. </span></strong><span style="font-family:"Calibri",sans-serif">Nevertheless, the KE can be measured using several well-established biochemical and analytical methods. Scientific confidence is highest when methods quantify specific lipid peroxidation products or oxidized lipid species directly, and lower when nonspecific colorimetric assays are used without appropriate controls or confirmatory methods.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Thiobarbituric acid reactive substances, often interpreted as MDA or MDA-like products</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Widely used and sensitive, but not fully specific because TBA can react with compounds other than MDA. Best used as a screening or comparative indicator of lipid peroxidation, particularly when supported by extraction, HPLC separation or additional markers (Buege and Aust, 1978; Ohkawa et al., 1979; Janero, 1990; Draper and Hadley, 1990).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">4-hydroxy-2-nonenal and related reactive aldehydes</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Mechanistically informative because 4-HNE is a major bioactive lipid peroxidation product. Antibody-based methods can detect protein adducts, whereas chromatographic or mass spectrometric methods improve specificity (Esterbauer et al., 1991; Uchida, 2003; Ayala et al., 2014).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Useful for detecting relatively early lipid peroxidation products. Hydroperoxides can be unstable and sample handling is critical. FOX-based methods provide a simple approach for lipid hydroperoxide detection (Jiang et al., 1992).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Chromatography and mass spectrometry</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">High specificity and quantitative power when standards and validated workflows are available. These methods can distinguish individual oxidized lipid species and are preferred for detailed mechanistic studies (Yin et al., 2011; Li et al., 2019).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Oxidation-sensitive fluorescent signal in cellular lipids</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Useful for cell-based or imaging applications and spatial localization, but probe specificity, photoxidation and calibration must be considered. Best used with complementary biochemical or analytical endpoints.</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<h4>References</h4>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP-Wiki. 2026. Key Event 1445: Increase, Lipid peroxidation. AOP-Wiki. Available at: https://aopwiki.org/events/1445. Accessed 14 May 2026.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Alam MR, Ehiguese FO, Vitale D, Martín-Díaz ML. 2022. Oxidative stress response to hydrogen peroxide exposure of Mytilus galloprovincialis and Ruditapes philippinarum: reduced embryogenesis success and altered biochemical response of sentinel marine bivalve species. Environmental Chemistry and Ecotoxicology 4:97-105.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ayala A, Munoz MF, Arguelles S. 2014. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 2014:360438. https://doi.org/10.1155/2014/360438.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Belaid C, Sbartai I. 2021. Assessing the effects of Thiram to oxidative stress responses in a freshwater bioindicator cladoceran (Daphnia magna). Chemosphere 268:128808. https://doi.org/10.1016/j.chemosphere.2020.128808.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Buege JA, Aust SD. 1978. Microsomal lipid peroxidation. Methods in Enzymology 52:302-310. https://doi.org/10.1016/S0076-6879(78)52032-6.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Cong B, Liu C, Wang L, Chai Y. 2020. The impact on antioxidant enzyme activity and related gene expression following adult zebrafish (Danio rerio) exposure to dimethyl phthalate. Animals 10(4):717. https://doi.org/10.3390/ani10040717.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Draper HH, Hadley M. 1990. Malondialdehyde determination as index of lipid peroxidation. Methods in Enzymology 186:421-431. https://doi.org/10.1016/0076-6879(90)86135-I.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Esperanza M, Cid A, Herrero C, Rioboo C. 2015. Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: screening cytotoxicity and genotoxicity endpoints. Aquatic Toxicology 165:210-221. https://doi.org/10.1016/j.aquatox.2015.06.004.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Esterbauer H, Schaur RJ, Zollner H. 1991. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biology and Medicine 11(1):81-128. https://doi.org/10.1016/0891-5849(91)90192-6.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Janero DR. 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biology and Medicine 9(6):515-540. https://doi.org/10.1016/0891-5849(90)90131-2.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Jiang ZY, Hunt JV, Wolff SP. 1992. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Analytical Biochemistry 202(2):384-389. https://doi.org/10.1016/0003-2697(92)90122-N.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Knauert S, Knauer K. 2008. The role of reactive oxygen species in copper toxicity to two freshwater green algae. Journal of Phycology 44(2):311-319. https://doi.org/10.1111/j.1529-8817.2008.00471.x.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Li L, Zhong S, Shen X, Li Q, Xu W, Tao Y, Yin H. 2019. Recent development on liquid chromatography-mass spectrometry analysis of oxidized lipids. Free Radical Biology and Medicine 144:16-34. https://doi.org/10.1016/j.freeradbiomed.2019.06.006.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Moore TD, Martin-Creuzburg D, Yampolsky LY. 2023. Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia. </span><span style="font-family:"Calibri",sans-serif">Oecologia 202(1):151-163. https://doi.org/10.1007/s00442-023-05382-1.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ohkawa H, Ohishi N, Yagi K. 1979. </span><span style="font-family:"Calibri",sans-serif">Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95(2):351-358. https://doi.org/10.1016/0003-2697(79)90738-3.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ouillon N, Sokolov EP, Otto S, Rehder G, Sokolova IM. 2021. Effects of variable oxygen regimes on mitochondrial bioenergetics and reactive oxygen species production in a marine bivalve, Mya arenaria. Journal of Experimental Biology 224(4):jeb237156. https://doi.org/10.1242/jeb.237156.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Tseng YC, Chen RD, Lucassen M, Schmidt MM, Dringen R, Abele D, Hwang PP. 2011. Exploring uncoupling proteins and antioxidant mechanisms under acute cold exposure in brains of fish. PLoS ONE 6(3):e18180. https://doi.org/10.1371/journal.pone.0018180.</span></span></span></p>
<p style="margin-left:24px"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Uchida K. 2003. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Progress in Lipid Research 42(4):318-343. https://doi.org/10.1016/S0163-7827(03)00014-6.</span></span></span></p>
<td><a href="/aops/267">Aop:267 - Uncoupling of oxidative phosphorylation leading to growth inhibition via glucose depletion</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/263">Aop:263 - Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/264">Aop:264 - Uncoupling of oxidative phosphorylation leading to growth inhibition via ATP depletion associated cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/265">Aop:265 - Uncoupling of oxidative phosphorylation leading to growth inhibition via increased cytosolic calcium</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/266">Aop:266 - Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased Na-K ATPase activity</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/268">Aop:268 - Uncoupling of oxidative phosphorylation leading to growth inhibition via mitochondrial swelling</a></td>
<td><a href="/aops/331">Aop:331 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/612">Aop:612 - Peroxisome proliferator-activated receptor alpha activation leading to early life stage mortality via reduced adenosine triphosphate</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/613">Aop:613 - Peroxisome proliferator-activated receptor alpha activation leading to early life stage mortality via increased reactive oxygen species production</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Reactive oxygen species leading to growth inhibition via protein oxidation and cell death</a></td>
<p style="text-align:justify">This key event is in general considered applicable to most eukaryotes, as the mitochondrion and oxidative phosphorylation are highly conserved (Roger 2017). <!--![endif]----></p>
<p style="text-align:justify">This key event is considered applicable to all life stages, as ATP synthesis by oxidative phosphorylation is an essential biological process for most living organisms.</p>
<p style="text-align:justify">This key event is considered sex-unspecific, as both males and females use oxidative phosphorylation as a main process to generate ATP.</p>
<p><!--![endif]----></p>
<h4>Key Event Description</h4>
<p style="text-align:justify">Decreased coupling of oxidative phosphorylation (OXPHOS), or uncoupling of OXPHOS, describes dissipation of protonmotive force (PMF) across the inner mitochondrial membrane (IMM) by environmental stressors. In eukaryotes, the mitochondrial electron transport chain mediates a series of redox reactions to create a PMF across the IMM. The PMF is used as energy to drive adenosine triphosphate (ATP) synthesis through phosphorylation of adenosine diphosphate (ADP). These processes are coupled and referred to as OXPHOS. A number of chemicals can dissipate the PMF, leading to uncoupling of OXPHOS. This key event describes the main outcome of the interactions between an uncoupler and the transmembrane PMF. An uncoupler can bind to a proton in the mitochondrial inter membrane space, transport the proton to the matrix side of the IMM, release the proton and move back to the inter membrane space. These processes are repeated until the transmembrane PMF is dissipated. This KE is therefore a lumped term of these processes and represents the final consequence of the interactions.</p>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify">Uncoupling of oxidative phosphorylation can be indicated by reduced mitochondrial membrane potential, increased proton leak and/or increased oxygen consumption rate.</p>
<ul>
<li>Mitochondrial membrane potential can be determined using ToxCast high-throughput screening bioassays such as “APR_HepG2_MitoMembPot”, “APR_Hepat_MitoFxnI”, and “APR_Mitochondrial_membrane_potential”, and the Tox21 high-throughput screening assay “tox21-mitotox-p1”.</li>
<li>Mitochondrial membrane potential can also be measured using commercially available fluorescent probes such as TMRM (tetramethylrhodamine, methyl ester, perchlorate), TMRE (tetramethylrhodamine, ethyl ester, perchlorate) and JC-1 (Perry 2011).</li>
<li>Proton leak and oxygen consumption rate can be measured using a high-resolution respirometry (Affourtit 2018) or a Seahorse XF analyzer (Divakaruni 2014).</li>
field-separator'></span><![endif]-->Affourtit C, Wong H-S, Brand MD. 2018. Measurement of proton leak in isolated mitochondria. In Palmeira CM, Moreno AJ, eds, <em>Mitochondrial Bioenergetics: Methods and Protocols</em>. Springer New York, New York, NY, pp 157-170.</p>
<p style="text-align:justify">Attene-Ramos MS, Huang R, Sakamuru S, Witt KL, Beeson GC, Shou L, Schnellmann RG, Beeson CC, Tice RR, Austin CP, Xia M. 2013. Systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening. <em>Chemical Research in Toxicology</em> 26:1323-1332. DOI: 10.1021/tx4001754.</p>
<p style="text-align:justify">Attene-Ramos MS, Huang RL, Michael S, Witt KL, Richard A, Tice RR, Simeonov A, Austin CP, Xia MH. 2015. Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease mitochondrial membrane potential. <em>Environ Health Persp</em> 123:49-56. DOI: 10.1289/ehp.1408642.</p>
<p style="text-align:justify">Divakaruni AS, Paradyse A, Ferrick DA, Murphy AN, Jastroch M. 2014. Chapter Sixteen - Analysis and Interpretation of Microplate-Based Oxygen Consumption and pH Data. In Murphy AN, Chan DC, eds, <em>Methods in Enzymology</em>. Vol 547. Academic Press, pp 309-354.</p>
<p style="text-align:justify">Escher BI, Schwarzenbach RP. 2002. Mechanistic studies on baseline toxicity and uncoupling of organic compounds as a basis for modeling effective membrane concentrations in aquatic organisms. <em>Aquatic Sciences</em> 64:20-35. DOI: 10.1007/s00027-002-8052-2.</p>
<p style="text-align:justify">Legradi J, Dahlberg A-K, Cenijn P, Marsh G, Asplund L, Bergman Å, Legler J. 2014. Disruption of Oxidative Phosphorylation (OXPHOS) by Hydroxylated Polybrominated Diphenyl Ethers (OH-PBDEs) Present in the Marine Environment. <em>Environmental Science & Technology</em> 48:14703-14711. DOI: 10.1021/es5039744.</p>
<p style="text-align:justify">Naven RT, Swiss R, Klug-Mcleod J, Will Y, Greene N. 2012. The development of structure-activity relationships for mitochondrial dysfunction: Uncoupling of oxidative phosphorylation. <em>Toxicol Sci</em> 131:271-278. DOI: 10.1093/toxsci/kfs279.</p>
<p style="text-align:justify">Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA. 2011. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. <em>BioTechniques</em> 50:98-115. DOI: 10.2144/000113610.</p>
<p style="text-align:justify">Roger AJ, Munoz-Gomez SA, Kamikawa R. 2017. The origin and diversification of mitochondria. <em>Curr Biol</em> 27:R1177-R1192. DOI: 10.1016/j.cub.2017.09.015.</p>
<p style="text-align:justify">Russom CL, Bradbury SP, Broderius SJ, Hammermeister DE, Drummond RA. 1997. Predicting modes of toxic action from chemical structure: Acute toxicity in the fathead minnow (Pimephales promelas). <em>Environ Toxicol Chem</em> 16:948-967. DOI: <a href="https://doi.org/10.1002/etc.5620160514">https://doi.org/10.1002/etc.5620160514</a>.</p>
<p style="text-align:justify">Shim J, Weatherly LM, Luc RH, Dorman MT, Neilson A, Ng R, Kim CH, Millard PJ, Gosse JA. 2016. Triclosan is a mitochondrial uncoupler in live zebrafish. <em>J Appl Toxicol</em> 36:1662-1667. DOI: 10.1002/jat.3311.</p>
<p style="text-align:justify">Sugiyama Y, Shudo T, Hosokawa S, Watanabe A, Nakano M, Kakizuka A. 2019. Emodin, as a mitochondrial uncoupler, induces strong decreases in adenosine triphosphate (ATP) levels and proliferation of B16F10 cells, owing to their poor glycolytic reserve. <em>Genes to Cells</em> 24:569-584. DOI: <a href="https://doi.org/10.1111/gtc.12712">https://doi.org/10.1111/gtc.12712</a>.</p>
<p style="text-align:justify">Terada H. 1990. Uncouplers of oxidative phosphorylation. <em>Environ Health Perspect</em> 87:213-218. DOI: 10.1289/ehp.9087213.</p>
<p style="text-align:justify">Troger F, Delp J, Funke M, van der Stel W, Colas C, Leist M, van de Water B, Ecker GF. 2020. Identification of mitochondrial toxicants by combined in silico and in vitro studies – A structure-based view on the adverse outcome pathway. <em>Computational Toxicology</em> 14:100123. DOI: <a href="https://doi.org/10.1016/j.comtox.2020.100123">https://doi.org/10.1016/j.comtox.2020.100123</a>.</p>
<p style="text-align:justify">Weatherly LM, Shim J, Hashmi HN, Kennedy RH, Hess ST, Gosse JA. 2016. Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes. <em>Journal of Applied Toxicology</em> 36:777-789. DOI: <a href="https://doi.org/10.1002/jat.3209">https://doi.org/10.1002/jat.3209</a>.</p>
<p style="text-align:justify">Xia M, Huang R, Shi Q, Boyd WA, Zhao J, Sun N, Rice JR, Dunlap PE, Hackstadt AJ, Bridge MF, Smith MV, Dai S, Zheng W, Chu PH, Gerhold D, Witt KL, DeVito M, Freedman JH, Austin CP, Houck KA, Thomas RS, Paules RS, Tice RR, Simeonov A. 2018. Comprehensive analyses and prioritization of Tox21 10K chemicals affecting mitochondrial function by in-depth mechanistic studies. <em>Environ Health Perspect</em> 126:077010. DOI: 10.1289/EHP2589.</p>
<td><a href="/aops/328">Aop:328 - Excessive reactive oxygen species production leading to mortality (2)</a></td>
<td>KeyEvent</td>
</tr>
<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/264">Aop:264 - Uncoupling of oxidative phosphorylation leading to growth inhibition via ATP depletion associated cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/263">Aop:263 - Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/290">Aop:290 - Mitochondrial ATP synthase antagonism leading to growth inhibition (1)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/291">Aop:291 - Mitochondrial ATP synthase antagonism leading to growth inhibition (2)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/286">Aop:286 - Mitochondrial complex III antagonism leading to growth inhibition (1)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/287">Aop:287 - Mitochondrial complex III antagonism leading to growth inhibition (2)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/266">Aop:266 - Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased Na-K ATPase activity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/612">Aop:612 - Peroxisome proliferator-activated receptor alpha activation leading to early life stage mortality via reduced adenosine triphosphate</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Reactive oxygen species leading to growth inhibition via protein oxidation and cell death</a></td>
<p style="text-align:justify">This key event is in general considered applicable to all eukaryotes utilizing ATP as a direct source of energy and signaling molecule.</p>
<p style="text-align:justify">This key event is considered applicable to all life stages, as all developmental stages require energy supply to maintain necessary physiological processes.</p>
<p style="text-align:justify">This key event is considered sex-unspecific, as both males and females use ATP as an essential energy molecule.</p>
<h4>Key Event Description</h4>
<p style="text-align:justify">Decreased adenosine triphosphate (ATP) pool describes the loss of balance between ATP synthesis and ATP consumption, leading to reduced total ATP. As a primary form of biological energy, ATP is used by many biological processes <!--[if supportFields]><span style='font-size:12.0pt;
ZH-CN;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]-->. Decrease in ATP level normally attributes to metabolic disorders in major ATP synthetic pathways, such as mitochondrial oxidative phosphorylation, fatty acid β-oxidation, glycolysis and plant photophosphorylation.</p>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify">-The ATP pool in cells or tissue can be quantified using a well-established ATP bioluminescent assay (Lemasters 1978; Wibom 1990). Assay principles: ATP can react with luciferase and luciferin from firefly and the luminescence emitted from the reaction is proportional to the ATP concentration: <!--![endif]----></p>
<p style="text-align:justify">-ToxCast high-throughput screening bioassays, such as “NCCT_HEK293T_CellTiterGLO” and “NIS_HEK293T_CTG_Cytotoxicity” can be used to measure this KE.</p>
field-separator'></span><![endif]-->Bonora M, Patergnani S, Rimessi A, De Marchi E, Suski JM, Bononi A, Giorgi C, Marchi S, Missiroli S, Poletti F, Wieckowski MR, Pinton P. 2012. ATP synthesis and storage. <em>Purinergic Signalling</em> 8:343-357. DOI: 10.1007/s11302-012-9305-8.</p>
<p>Lemasters JJ, Hackenbrock CR. 1978. [4] Firefly luciferase assay for ATP production by mitochondria. <em>Methods in Enzymology</em>. Vol 57. Academic Press, pp 36-50.</p>
<p>Wibom R, Lundin A, Hultman E. 1990. A sensitive method for measuring ATP-formation in rat muscle mitochondria. <em>Scandinavian Journal of Clinical and Laboratory Investigation</em> 50:143-152. DOI: 10.1080/00365519009089146.</p>
<td><a href="/aops/502">Aop:502 - Decrease, cholesterol synthesis leads to orofacial clefting</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/591">Aop:591 - DBDPE-induced DNA damage increase in liver leading to Non-alcoholic fatty liver disease via liver steatosis and inhibition of regeneration</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell cycle disruption</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<p>This key event is in general applicable to all life stages. As cell proliferation not only occurs in developing organisms, but also in adults.</p>
<p> </p>
<p><strong>Sex applicability domain</strong></p>
<p>This key event is sex-unspecific, as both genders use the same cell proliferation mechanisms.</p>
<h4>Key Event Description</h4>
<p style="text-align:justify">Decreased cell proliferation describes the outcome of reduced cell division and cell growth. Cell proliferation is considered the main mechanism of tissue and organismal growth (Conlon 1999). Decreased cell proliferation has been associated with abnormal growth-factor signaling and cellular energy depletion (DeBerardinis 2008).</p>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify">Multiple types of <em>in vitro</em> bioassays can be used to measure this key event:</p>
<ul>
<li>ToxCast high-throughput screening bioassays such as “BSK_3C_Proliferation”, “BSK_CASM3C_Proliferation” and “BSK_SAg_Proliferation” can be used to measure cell proliferation status.</li>
<li>Commercially available methods such as the well-established 5-bromo-2’-deoxyuridine (BrdU) (Raza 1985; Muir 1990) or 5-ethynyl-2’-deoxyuridine (EdU) assay. Both assays measure DNA synthesis in dividing cells to indicate proliferation status.<!--![endif]----></li>
</ul>
<h4>References</h4>
<p style="text-align:justify">Conlon I, Raff M. 1999. Size control in animal development. <em>Cell</em> 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.</p>
<p style="text-align:justify">DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. 2008. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. <em>Cell Metabolism</em> 7:11-20. DOI: <a href="https://doi.org/10.1016/j.cmet.2007.10.002">https://doi.org/10.1016/j.cmet.2007.10.002</a>.</p>
<p style="text-align:justify">Muir D, Varon S, Manthorpe M. 1990. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures. <em>Analytical Biochemistry</em> 185:377-382. DOI: <a href="https://doi.org/10.1016/0003-2697(90)90310-6">https://doi.org/10.1016/0003-2697(90)90310-6</a>.</p>
<p style="text-align:justify">Raza A, Spiridonidis C, Ucar K, Mayers G, Bankert R, Preisler HD. 1985. Double labeling of S-phase murine cells with bromodeoxyuridine and a second DNA-specific probe. <em>Cancer Research</em> 45:2283-2287.</p>
<td><a href="/aops/263">Aop:263 - Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/290">Aop:290 - Mitochondrial ATP synthase antagonism leading to growth inhibition (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/291">Aop:291 - Mitochondrial ATP synthase antagonism leading to growth inhibition (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/286">Aop:286 - Mitochondrial complex III antagonism leading to growth inhibition (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/287">Aop:287 - Mitochondrial complex III antagonism leading to growth inhibition (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/245">Aop:245 - Reduction in photophosphorylation leading to growth inhibition in aquatic plants</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/265">Aop:265 - Uncoupling of oxidative phosphorylation leading to growth inhibition via increased cytosolic calcium</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/264">Aop:264 - Uncoupling of oxidative phosphorylation leading to growth inhibition via ATP depletion associated cell death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/266">Aop:266 - Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased Na-K ATPase activity</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/267">Aop:267 - Uncoupling of oxidative phosphorylation leading to growth inhibition via glucose depletion</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/268">Aop:268 - Uncoupling of oxidative phosphorylation leading to growth inhibition via mitochondrial swelling</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/473">Aop:473 - Energy deposition from internalized Ra-226 decay lower oxygen binding capacity of hemocyanin</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/567">Aop:567 - Binding to plastoquinone B site leading to decreased population growth rate via photosystem II inhibition</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell cycle disruption</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Reactive oxygen species leading to growth inhibition via protein oxidation and cell death</a></td>
<p style="text-align:justify">This key event is sex-unspecific.</p>
<h4>Key Event Description</h4>
<p style="text-align:justify">Decreased growth refers to a reduction in size and/or weight of a tissue, organ or individual organism. Growth is normally controlled by growth factors and mainly achieved through cell proliferation (Conlon 1999).</p>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify">Growth can be indicated by measuring weight, length, total volume, and/or total area of a tissue, organ or individual organism. </p>
<h4>Regulatory Significance of the AO</h4>
<p style="text-align:justify">Growth is a regulatory relevant chronic toxicity endpoint for almost all organisms. Multiple OECD test guidelines have included growth either as a main endpoint of concern, or as an additional endpoint to be considered in the toxicity assessments. Relevant test guidelines include, but not only limited to:</p>
<p style="text-align:justify"> </p>
<p>-Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test</p>
<p>-Test No. 228: Determination of Developmental Toxicity to Dipteran Dung Flies (Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae))</p>
<p>-Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA)</p>
<p>-Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents</p>
<p>-Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents</p>
style='mso-element:field-separator'></span><![endif]-->Conlon I, Raff M. 1999. Size control in animal development. <em>Cell</em> 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.</p>
<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>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/521">Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/186">unknown MIE leading to renal failure and mortality</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/497">ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/540">Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>adjacent</td>
<td>High</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/aops/462">Activation of reactive oxygen species leading the atherosclerosis</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/396">Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/26">Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/534">Succinate dehydrogenase (SDH) inhibition leads to oxidative stress</a></td>
<td><a href="/aops/596">Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/599">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/600">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/601">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/602">Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/603">Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/472">DNA adduct formation leading to kidney failure</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/324">Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell cycle disruption</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/325">Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell death</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/326">Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/332">Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/333">Reactive oxygen species leading to growth inhibition via protein oxidation and cell death</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This KER is broadly applicable to aerobic eukaryotic systems in which ROS production and antioxidant buffering can be measured. The current AOP-Wiki relationship page identifies human, mouse and rat with high evidence, but the ROS-growth evidence base supports extension to algae, fish, crustaceans, mollusks and other organisms relevant to environmental toxicology (AOP-Wiki, 2026a). The relationship is expected to be conserved because it is based on redox chemistry and conserved antioxidant-defense systems rather than on a taxon-specific receptor or signaling pathway.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The applicability domain should nevertheless be bounded by biological context and measurement feasibility. This KER is most relevant when the upstream KE is a measurable increase in ROS and the downstream KE is a measurable redox imbalance or antioxidant-response state rather than a distal oxidative damage endpoint alone. In organisms or compartments where ROS cannot be measured directly, evidence may rely on antioxidant-response or oxidative damage biomarkers, but these should be interpreted as indirect support. Applicability is strongest when ROS and oxidative stress endpoints are measured in the same system under the same exposure conditions.</span></span></span></p>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This KER describes the causal and predictive relationship by which an increase in reactive oxygen species leads to oxidative stress. ROS include superoxide, hydrogen peroxide, hydroxyl radical and secondary oxygen-derived reactive products. At low or transient levels, ROS can participate in normal cell signaling. However, when ROS production, flux or local concentration exceeds the capacity of enzymatic and non-enzymatic antioxidant defenses, the redox balance of the biological system shifts toward an oxidizing state, producing oxidative stress (Schieber and Chandel, 2014; Sies et al., 2017).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"> <span style="font-family:"Calibri",sans-serif">The downstream KE, oxidative stress, is not identical to increased ROS. Rather, it represents a systems-level imbalance between pro-oxidant pressure and antioxidant or repair capacity. The KER therefore depends not only on the magnitude of ROS increase, but also on the duration, localization and chemical identity of the ROS, the capacity of scavenging systems such as glutathione, superoxide dismutase, catalase and glutathione peroxidases, and the ability of the cell or organism to activate adaptive redox responses such as NRF2 signaling (Halliwell and Gutteridge, 2015; Griendling et al., 2016; Sies et al., 2017).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Within the ROS-growth AOP network, Relationship 2009 functions as a shared upstream KER. It connects the early measurable perturbation of increased ROS to the central hub event of oxidative stress, from which downstream AOP branches proceed through oxidative DNA damage, lipid peroxidation, protein oxidation, mitochondrial dysfunction, ATP depletion, altered cell proliferation, cell injury/death and decreased growth. This KER should remain modular and stressor-agnostic; stressor-specific mechanisms of ROS generation should be described in MIE or stressor sections where appropriate.</span></span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Biological plausibility of Relationship 2009 is high. ROS are produced endogenously by mitochondrial electron transport, oxidase enzymes, peroxisomal reactions, photosynthetic electron transport and immune-cell oxidant systems, and they may also be generated by redox-cycling chemicals, metals, radiation and other stressors (Bedard and Krause, 2007; Murphy, 2009; Halliwell and Gutteridge, 2015). Oxidative stress is defined as a disturbance in the balance between oxidants and antioxidants in favor of oxidants, leading to disruption of redox signaling and/or molecular damage (Sies et al., 2017). Therefore, a sufficient increase in ROS has a direct mechanistic basis for causing oxidative stress when antioxidant and repair capacity are exceeded.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This relationship is also strongly supported by the known biology of antioxidant defenses. Superoxide dismutases convert superoxide to hydrogen peroxide; catalase, glutathione peroxidases and peroxiredoxins reduce hydrogen peroxide and organic peroxides; and glutathione and thioredoxin systems maintain protein thiol redox balance. Increased ROS can consume these defenses, oxidize redox-sensitive proteins, activate NRF2-dependent antioxidant response pathways, and produce oxidative modification of lipids, proteins and nucleic acids (Schieber and Chandel, 2014; Griendling et al., 2016; Sies et al., 2017).</span></span></span></p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Empirical support for this KER is high. Numerous studies across taxa and stressor classes demonstrate concordant increases in ROS or ROS-generating conditions and oxidative stress endpoints. The strongest evidence comes from studies measuring both ROS and antioxidant-response or oxidative-stress biomarkers in the same biological system. Several examples from the ROS-growth concordance table are summarized below.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">DCFH-DA fluorescence increased; LOEC for ROS approximately 0.5 uM paraquat.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">SOD, POD and CAT activities increased at similar concentrations; antioxidant enzymes were approximately 3-5-fold above control at 0.5 uM.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Dose concordance supports ROS increase leading to oxidative stress in a photosynthetic eukaryote.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">ROS occurs at lower or similar concentrations than antioxidant and damage markers, supporting dose concordance.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">ROS increased early, with maximum response around 6 h.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Antioxidant enzyme activities and antioxidant gene expression changed following the ROS response.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">ROS increased at the lowest tested dose by day 42.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Concordant ROS and antioxidant-response changes support the relationship in mammals.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Stressor is thiol-reactive and associated with oxidative challenge; direct ROS was not the primary endpoint.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Supports downstream oxidative stress following a stressor known to disturb redox balance; direct ROS evidence is weaker than in rows with ROS measurement.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Hypoxia/reoxygenation is a recognized ROS-generating condition in mitochondria.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ouillon et al. (2021)</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The main uncertainties relate to measurement specificity and context dependence. ROS are chemically diverse and often short-lived, so different assays may detect different ROS species or generalized oxidant-dependent probe oxidation rather than a single ROS concentration. DCFH-DA and related probes are useful screening tools but can be influenced by peroxidases, metals, light, probe loading and cellular esterase activity (Wardman, 2007; Kalyanaraman et al., 2012). Consequently, apparent ROS increases must be interpreted with assay limitations in mind.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">A second uncertainty is that ROS increases are not always adverse. Transient or localized ROS signals may activate adaptive stress responses and restore redox homeostasis without producing sustained oxidative stress. Conversely, oxidative stress may be inferred from antioxidant enzyme induction or oxidative damage biomarkers in studies where ROS were not directly measured. These cases support the KER less strongly than studies with direct, temporally resolved ROS measurements. Differences among taxa, life stages, tissues, exposure durations and antioxidant capacities may alter the threshold at which increased ROS becomes oxidative stress.</span></span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Quantitative understanding of this KER is low to moderate. The qualitative relationship is well established: oxidative stress occurs when ROS production or flux exceeds antioxidant and repair capacity. However, a universal quantitative threshold for ROS leading to oxidative stress cannot be defined because the relationship depends strongly on ROS species, subcellular localization, measurement method, antioxidant capacity, exposure duration, organism, cell type and co-stressors (Kalyanaraman et al., 2012; Griendling et al., 2016; Sies et al., 2017).</span></span></span></p>
<strong>Response-response relationship</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Response-response information is available in specific systems. For example, in Chlorella vulgaris exposed to paraquat, ROS and antioxidant enzyme responses were observed at approximately 0.5 uM after 24 h, indicating local dose concordance between the upstream and downstream events (Qian et al., 2009). In Daphnia magna exposed to paraquat, ROS induction was reported at lower concentrations than antioxidant enzyme and TBARS responses, supporting an expected dose sequence in which ROS increases precede oxidative stress endpoints (Barata et al., 2005). These examples provide semi-quantitative support, but they cannot be generalized across all taxa or stressors.</span></span></span></p>
<strong>Time-scale</strong>
<p><span style="font-size:18px"><span style="font-family:"Calibri",sans-serif">The time scale of the KER can range from minutes to hours for ROS-sensitive signaling and antioxidant pathway activation, and from hours to days for measurable changes in antioxidant enzyme activities, glutathione status or oxidative damage biomarkers. In pathogen-exposed golden pompano, ROS increased early, followed by antioxidant enzyme and gene expression responses over subsequent hours to days, supporting temporal concordance (Gao et al., 2022).</span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><strong><span style="font-family:"Calibri",sans-serif"><span style="color:black">Effect on the KER</span></span></strong></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Levels and activities of GSH, SOD, CAT, GPx, peroxiredoxins, thioredoxin systems and antioxidant vitamins.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Higher antioxidant capacity buffers ROS and raises the threshold for oxidative stress; depleted or impaired antioxidant systems lower the threshold.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Induction of antioxidant and detoxification genes through NRF2-dependent signaling.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Adaptive NRF2 activation may reduce progression from increased ROS to sustained oxidative stress, but strong NRF2 activation can also serve as evidence that ROS has perturbed redox homeostasis.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Mitochondria, chloroplasts, peroxisomes, membranes, nuclei and phagosomes differ in ROS production and local antioxidant buffering.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Localized ROS production can cause oxidative stress in a specific compartment even when whole-cell ROS measurements are modest.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Acute pulses, chronic low-level exposure and repeated stress can produce different redox outcomes.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Short pulses may be buffered or adaptive; sustained or repeated ROS elevations increase the probability of oxidative stress.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Oxygen tension affects mitochondrial electron transport and ROS formation.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Reoxygenation after hypoxia can increase mitochondrial ROS and enhance oxidative stress.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Temperature and metabolic demand alter oxygen flux, mitochondrial activity and antioxidant capacity.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Higher metabolic activity or thermal stress can increase ROS formation and shift the balance toward oxidative stress.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Stressor type influences the ROS species, localization, time course and threshold for oxidative stress.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Bedard and Krause (2007); Murphy (2009); Qian et al. (2009); Gao et al. (2022).</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Known feedback and feedforward mechanisms influence the linkage. NRF2-dependent antioxidant responses can reduce ROS and restore homeostasis, whereas mitochondrial dysfunction, lipid peroxidation, inflammation and redox-sensitive signaling can amplify ROS generation and sustain oxidative stress. These feedbacks make the KER dynamic and nonlinear, particularly under chronic exposure or repeated stress.</span></span></span></p>
<h4>References</h4>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP-Wiki. 2026a. Relationship 2009: Increase, ROS leads to Increase, Oxidative stress. AOP-Wiki. Available at: https://aopwiki.org/relationships/2009. Accessed 14 May 2026.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">AOP-Wiki. 2026c. Event 1392: Increase, Oxidative stress. AOP-Wiki. Available at: https://aopwiki.org/events/1392. Accessed 14 May 2026.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Barata C, Varo I, Navarro JC, Arun S, Porte C. 2005. Antioxidant enzyme activities and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox cycling compounds. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 140(2):175-186. https://doi.org/10.1016/j.cca.2005.01.013.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Bedard K, Krause KH. 2007. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological Reviews 87(1):245-313. https://doi.org/10.1152/physrev.00044.2005.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Dickinson BC, Chang CJ. 2011. Chemistry and biology of reactive oxygen species in signaling or stress responses. Nature Chemical Biology 7(8):504-511. https://doi.org/10.1038/nchembio.607.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Esperanza M, Cid A, Herrero C, Rioboo C. 2015. Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: screening cytotoxicity and genotoxicity endpoints. Aquatic Toxicology 165:210-221. https://doi.org/10.1016/j.aquatox.2015.06.004.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Gao J, Liu M, Guo H, Zhu K, Liu B, Liu B, Zhang N, Sun X, Jiang S, Zhang D. 2022. ROS induced by Streptococcus agalactiae activate inflammatory responses via the TNF-alpha/NF-kappaB signaling pathway in golden pompano Trachinotus ovatus (Linnaeus, 1758). Antioxidants 11(9):1809. https://doi.org/10.3390/antiox11091809.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Griendling KK, Touyz RM, Zweier JL, Dikalov S, Chilian W, Chen YR, Harrison DG, Bhatnagar A. 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-e75. https://doi.org/10.1161/RES.0000000000000110.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Halliwell B, Gutteridge JMC. 2015. Free Radicals in Biology and Medicine. 5th ed. Oxford: Oxford University Press.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Haque MN, Eom HJ, Nam SE, Shin YK, Rhee JS. 2019. Chlorothalonil induces oxidative stress and reduces enzymatic activities of Na+/K+-ATPase and acetylcholinesterase in gill tissues of marine bivalves. PLoS ONE 14(4):e0214236. https://doi.org/10.1371/journal.pone.0214236.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Jian Z, Guo H, Liu H, Cui H, Fang J, Zuo Z, Deng J, Li Y, Wang X, Zhao L. 2020. Oxidative stress, apoptosis and inflammatory responses involved in copper-induced pulmonary toxicity in mice. Aging 12(17):16867-16886. https://doi.org/10.18632/aging.103585.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Kalyanaraman B, Darley-Usmar V, Davies KJA, Dennery PA, Forman HJ, Grisham MB, Mann GE, Moore K, Roberts LJ II, Ischiropoulos H. 2012. Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radical Biology and Medicine 52(1):1-6. https://doi.org/10.1016/j.freeradbiomed.2011.09.030.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Murphy MP. 2009. How mitochondria produce reactive oxygen species. Biochemical Journal 417(1):1-13. https://doi.org/10.1042/BJ20081386.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ouillon N, Sokolov EP, Otto S, Rehder G, Sokolova IM. 2021. Effects of variable oxygen regimes on mitochondrial bioenergetics and reactive oxygen species production in a marine bivalve, Mya arenaria. Journal of Experimental Biology 224(4):jeb237156. https://doi.org/10.1242/jeb.237156.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Pan YX, Luo Z, Zhuo MQ, Wei CC, Chen GH, Song YF. 2018. Oxidative stress and mitochondrial dysfunction mediated Cd-induced hepatic lipid accumulation in zebrafish Danio rerio. Aquatic Toxicology 199:12-20. https://doi.org/10.1016/j.aquatox.2018.03.017.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Qian H, Chen W, Sun L, Jin Y, Liu W, Fu Z. 2009. Inhibitory effects of paraquat on photosynthesis and the response to oxidative stress in Chlorella vulgaris. Ecotoxicology 18(5):537-543. https://doi.org/10.1007/s10646-009-0311-8.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Schieber M, Chandel NS. 2014. ROS function in redox signaling and oxidative stress. Current Biology 24(10):R453-R462. https://doi.org/10.1016/j.cub.2014.03.034.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Sies H, Berndt C, Jones DP. 2017. Oxidative stress. Annual Review of Biochemistry 86:715-748. https://doi.org/10.1146/annurev-biochem-061516-045037.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Tseng YC, Chen RD, Lucassen M, Schmidt MM, Dringen R, Abele D, Hwang PP. 2011. Exploring uncoupling proteins and antioxidant mechanisms under acute cold exposure in brains of fish. PLoS ONE 6(3):e18180. https://doi.org/10.1371/journal.pone.0018180.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Wardman P. 2007. Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radical Biology and Medicine 43(7):995-1022. https://doi.org/10.1016/j.freeradbiomed.2007.06.026.</span></span></span></p>
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<h4><a href="/relationships/3116">Relationship: 3116: Increase, Oxidative Stress leads to Increase, LPO</a></h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This KER is broadly applicable to aerobic biological systems containing oxidizable lipids. It is particularly relevant to membranes and lipid-rich tissues or compartments, including plasma membranes, mitochondrial membranes, chloroplast membranes, digestive gland, liver, nervous tissue and reproductive tissues. The relationship is expected to be conserved across taxa because it is based on fundamental redox chemistry and lipid radical chain reactions rather than on a taxon-specific receptor pathway.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The KER should be applied most confidently when both upstream oxidative stress and downstream lipid peroxidation are measured under the same exposure conditions. Applicability is strongest when oxidative stress is assessed by redox imbalance or antioxidant-response endpoints and lipid peroxidation is measured using specific markers such as MDA, 4-HNE or lipid hydroperoxides. Applicability is weaker when lipid peroxidation is inferred solely from nonspecific TBARS responses without supporting oxidative-stress biomarkers or when the exposure context is dominated by physical membrane disruption rather than redox-mediated chemistry.</span></span></span></p>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This KER describes the relationship by which an increase in oxidative stress leads to an increase in lipid peroxidation. Oxidative stress represents a shift toward a pro-oxidant state in which reactive oxygen species, reactive nitrogen species, redox-active intermediates, or weakened antioxidant defenses exceed the buffering capacity of the biological system. Lipid peroxidation is a chain reaction in which oxidants abstract hydrogen atoms from susceptible lipids, particularly polyunsaturated fatty acids, producing lipid radicals, lipid peroxyl radicals, lipid hydroperoxides and secondary reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) (Halliwell and Gutteridge, 2015; Ayala et al., 2014; Yin et al., 2011).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The relationship is biologically plausible because increased oxidative pressure raises the probability of radical initiation and propagation in lipid-rich compartments, especially biological membranes. Once initiated, lipid peroxidation can propagate through neighboring lipids and can be amplified by transition metals, oxygen availability, membrane composition and reduced antioxidant protection. The downstream KE therefore reflects a measurable chemical and biological consequence of upstream oxidative stress rather than a separate stressor-specific mechanism. The KER is modular and can be reused wherever oxidative stress is followed by measurable increases in lipid oxidation products.</span></span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Biological plausibility of this KER is high. The mechanistic basis is well established in chemistry and biology: oxidative stress increases reactive species capable of initiating lipid radical formation, and lipid radicals propagate chain reactions that generate lipid hydroperoxides and reactive aldehydes (Halliwell and Gutteridge, 2015; Ayala et al., 2014; Yin et al., 2011). Polyunsaturated fatty acids are particularly susceptible because bis-allylic hydrogens are readily abstracted, making membrane lipid composition a major determinant of sensitivity. Endogenous antioxidant systems, including glutathione peroxidases, peroxiredoxins, vitamin E, glutathione and other radical-scavenging systems, normally limit lipid peroxidation. When oxidative stress overwhelms these defenses, lipid peroxidation increases.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The structural and functional relationship between the two KEs is direct: the upstream KE increases the chemical conditions that initiate and propagate the downstream lipid oxidation process. This relationship is broadly accepted across toxicology, cell biology, physiology and environmental stress biology.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Paraquat increased ROS and induced antioxidant enzymes; ROS and oxidative stress responses were observed at concentrations that support a pro-oxidant state leading to downstream oxidative damage.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Supports upstream oxidative-stress induction by a redox-cycling herbicide and provides context for lipid-damage progression in algae.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">SOD/CAT induction occurred with increased MDA/TBARS, with MDA elevated at similar or higher concentrations than antioxidant-response markers.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Supports dose concordance between oxidative stress biomarkers and lipid peroxidation in freshwater green microalgae.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Significant TBARS/MDA increase occurred at >=0.5 uM paraquat, with associated mitochondrial depolarization at similar concentrations.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Supports oxidative-stress-driven lipid peroxidation following exposure to a superoxide-generating herbicide.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">ROS induction was observed at lower concentrations, followed by antioxidant enzyme induction and TBARS responses at higher concentrations.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">GSH depletion and increased MDA/TBARS were observed after thiram exposure.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Supports empirical linkage between redox imbalance and lipid peroxidation in a freshwater crustacean.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Supports the role of lipid composition as a modulator and provides evidence that increased lipid susceptibility enhances peroxidation and downstream mitochondrial effects.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Antioxidant enzyme changes and MDA increases were observed after exposure.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Supports concordance between oxidative-stress biomarkers and lipid peroxidation in fish.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Hydrogen peroxide, 21 d or 48 h</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Antioxidant enzyme activation was observed at lower concentrations than lipid peroxidation in digestive gland.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Supports dose concordance between direct oxidant exposure, oxidative-stress response and lipid peroxidation in bivalves.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Sustained H2O2 production increased MDA at higher production rates.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Montserrat-Mesquida et al. (2024).</span></span></span></p>
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<strong>Uncertainties and Inconsistencies</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The overall evidence for this KER is strong, but several uncertainties influence interpretation. First, lipid peroxidation biomarkers can be nonspecific or method-dependent. TBARS is widely used but can overestimate MDA or respond to non-lipid-derived substances; more specific methods such as HPLC, LC-MS/MS or measurement of 4-HNE and lipid hydroperoxides provide stronger evidence (Ayala et al., 2014; Yin et al., 2011). Second, oxidative stress is often inferred from antioxidant enzyme induction or glutathione perturbation rather than directly measured ROS flux. Third, lipid peroxidation depends strongly on membrane lipid composition, antioxidant status, metal availability and exposure duration, so the same oxidative-stress magnitude may not produce the same lipid peroxidation response in all systems. Finally, adaptive antioxidant responses may delay or suppress lipid peroxidation after mild oxidative stress, creating apparent temporal or dose-response discordance in some studies.</span></span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Quantitative understanding of this KER is moderate. The qualitative and mechanistic relationship is well established, but a universal quantitative threshold for oxidative stress leading to lipid peroxidation cannot be defined because the response depends on lipid composition, antioxidant capacity, oxygen availability, transition metals, exposure duration, stressor chemistry and assay method (Ayala et al., 2014; Yin et al., 2011; Sies et al., 2017).</span></span></span><span style="font-size:18px"><span style="font-family:Cambria,serif"> </span></span></p>
<strong>Response-response relationship</strong>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Response-response evidence exists in specific systems. In green microalgae exposed to copper, antioxidant enzyme induction and MDA/TBARS increases occurred over the same concentration range, supporting dose concordance (Knauert and Knauer, 2008). In Daphnia magna exposed to paraquat, ROS induction occurred at lower concentrations than antioxidant enzyme and TBARS responses, suggesting that increased ROS and oxidative stress precede lipid peroxidation (Barata et al., 2005). In bivalves exposed to hydrogen peroxide, antioxidant enzyme activation occurred at lower concentrations than lipid peroxidation in digestive gland, also supporting a staged relationship (Alam et al., 2022).</span></span></span></p>
<strong>Time-scale</strong>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The time scale of the linkage can range from minutes to days. Chemical initiation of lipid radicals can occur rapidly when reactive species are present, but commonly measured endpoints such as MDA, TBARS, 4-HNE or lipid hydroperoxides often become detectable over hours to days depending on exposure intensity and tissue antioxidant capacity. Quantitative prediction of lipid peroxidation from oxidative-stress measurements therefore remains system-specific and is best supported when both KEs are measured in the same biological context and time course.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><strong><span style="font-family:"Calibri",sans-serif"><span style="color:black">Effect on the KER</span></span></strong></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Increases the probability and magnitude of lipid peroxidation for a given oxidative-stress level.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ayala et al. (2014); Yin et al. (2011); Moore et al. (2023).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Includes glutathione, glutathione peroxidases, catalase, peroxiredoxins, vitamin E and other lipid-soluble antioxidants.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Halliwell and Gutteridge (2015); Sies et al. (2017); Belaid and Sbartai (2021).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Enhances initiation and propagation of lipid peroxidation, often lowering the threshold for the downstream KE.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Halliwell and Gutteridge (2015); Knauert and Knauer (2008); Regoli and Giuliani (2014).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">May increase ROS production and alter membrane susceptibility to lipid peroxidation.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">TBARS, MDA, 4-HNE and lipid hydroperoxide methods differ in specificity and kinetics.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ayala et al. (2014); Yin et al. (2011).</span></span></span></p>
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<h4>References</h4>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Alam MR, Ehiguese FO, Vitale D, Martín-Díaz ML. 2022. Oxidative stress response to hydrogen peroxide exposure of Mytilus galloprovincialis and Ruditapes philippinarum: reduced embryogenesis success and altered biochemical response of sentinel marine bivalve species. Environmental Chemistry and Ecotoxicology 4:97-105. https://doi.org/10.1016/j.enceco.2022.01.002.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Almaida-Pagán PF, Lucas-Sánchez A, Tocher DR. 2014. Changes in mitochondrial membrane composition and oxidative status during rapid growth, maturation and aging in zebrafish, Danio rerio. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 1841(7):1003-1011. https://doi.org/10.1016/j.bbalip.2014.04.004.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ayala A, Munoz MF, Arguelles S. 2014. </span><span style="font-family:"Calibri",sans-serif">Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 2014:360438. https://doi.org/10.1155/2014/360438.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Barata C, Varo I, Navarro JC, Arun S, Porte C. 2005. Antioxidant enzyme activities and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox cycling compounds. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 140(2):175-186. https://doi.org/10.1016/j.cca.2005.01.013.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Belaid C, Sbartai I. 2021. Assessing the effects of thiram to oxidative stress responses in a freshwater bioindicator cladoceran (Daphnia magna). Chemosphere 268:128808. https://doi.org/10.1016/j.chemosphere.2020.128808.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Cong B, Liu C, Wang L, Chai Y. 2020. The impact on antioxidant enzyme activity and related gene expression following adult zebrafish (Danio rerio) exposure to dimethyl phthalate. Animals 10(4):717. https://doi.org/10.3390/ani10040717.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Esperanza M, Cid A, Herrero C, Rioboo C. 2015. Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: screening cytotoxicity and genotoxicity endpoints. Aquatic Toxicology 165:210-221. https://doi.org/10.1016/j.aquatox.2015.06.004.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Halliwell B, Gutteridge JMC. 2015. Free Radicals in Biology and Medicine. 5th ed. Oxford: Oxford University Press.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Haque MN, Eom HJ, Nam SE, Shin YK, Rhee JS. 2019. Chlorothalonil induces oxidative stress and reduces enzymatic activities of Na+/K+-ATPase and acetylcholinesterase in gill tissues of marine bivalves. PLoS ONE 14(4):e0214236. https://doi.org/10.1371/journal.pone.0214236.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Knauert S, Knauer K. 2008. The role of reactive oxygen species in copper toxicity to two freshwater green algae. Journal of Phycology 44(2):311-321. https://doi.org/10.1111/j.1529-8817.2008.00471.x.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Montserrat-Mesquida M, Ferrer MD, Pons A, Sureda A, Capó X. 2024. Effects of chronic hydrogen peroxide exposure on mitochondrial oxidative stress genes, ROS production and lipid peroxidation in HL60 cells. Mitochondrion 76:101869. https://doi.org/10.1016/j.mito.2024.101869.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Moore TD, Martin-Creuzburg D, Yampolsky LY. 2023. Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia. Oecologia 202(1):151-163. https://doi.org/10.1007/s00442-023-05382-1.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ouillon N, Sokolov EP, Otto S, Rehder G, Sokolova IM. 2021. Effects of variable oxygen regimes on mitochondrial bioenergetics and reactive oxygen species production in a marine bivalve, Mya arenaria. Journal of Experimental Biology 224(4):jeb237156. https://doi.org/10.1242/jeb.237156.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Pan YX, Luo Z, Zhuo MQ, Wei CC, Chen GH, Song YF. 2018. Oxidative stress and mitochondrial dysfunction mediated Cd-induced hepatic lipid accumulation in zebrafish Danio rerio. Aquatic Toxicology 199:12-20. https://doi.org/10.1016/j.aquatox.2018.03.017.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Qian H, Chen W, Sun L, Jin Y, Liu W, Fu Z. 2009. Inhibitory effects of paraquat on photosynthesis and the response to oxidative stress in Chlorella vulgaris. Ecotoxicology 18(5):537-543. https://doi.org/10.1007/s10646-009-0311-8.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Regoli F, Giuliani ME. 2014. Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms. Marine Environmental Research 93:106-117. https://doi.org/10.1016/j.marenvres.2013.07.006.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Schieber M, Chandel NS. 2014. ROS function in redox signaling and oxidative stress. Current Biology 24(10):R453-R462. https://doi.org/10.1016/j.cub.2014.03.034.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Sies H, Berndt C, Jones DP. 2017. Oxidative stress. Annual Review of Biochemistry 86:715-748. https://doi.org/10.1146/annurev-biochem-061516-045037.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Sokolov EP, Markert S, Hinzke T, Hirschfeld C, Becher D, Ponsuksili S, Sokolova IM. 2019. Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas. </span><span style="font-family:"Calibri",sans-serif">Journal of Proteomics 194:99-111. https://doi.org/10.1016/j.jprot.2018.12.009.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Tseng YC, Chen RD, Lucassen M, Schmidt MM, Dringen R, Abele D, Hwang PP. 2011. </span><span style="font-family:"Calibri",sans-serif">Exploring uncoupling proteins and antioxidant mechanisms under acute cold exposure in brains of fish. </span><span style="font-family:"Calibri",sans-serif">PLoS ONE 6(3):e18180. https://doi.org/10.1371/journal.pone.0018180.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This KER is applicable to aerobic eukaryotic systems with functional mitochondria and oxidizable membrane lipids. The relationship is especially relevant to biological contexts where mitochondrial membranes are enriched in cardiolipin and other polyunsaturated lipids, where oxidative stress targets membrane compartments, or where environmental conditions promote ROS formation and lipid radical propagation. The KER is likely most useful for stressors that induce oxidative membrane damage, including redox cycling chemicals, metals, radiation, hypoxia/reoxygenation, temperature stress, mitochondrial toxicants, and inflammatory or endogenous ROS-generating conditions.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The KER should be applied with greatest confidence when lipid peroxidation is measured using specific or well-characterized markers and when the downstream mitochondrial event is assessed by direct coupling-related endpoints such as respiratory control ratio, proton leak, OXPHOS coupling efficiency, mitochondrial membrane potential, or state 3/state 4 respiration. Applicability is less certain when lipid peroxidation is measured only at the whole-organism level without compartmental resolution, or when decreased coupling is caused primarily by direct uncouplers or respiratory-chain inhibitors without evidence of lipid oxidative damage.</span></span></span></p>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">This KER describes the relationship by which increased lipid peroxidation leads to decreased coupling of oxidative phosphorylation. Lipid peroxidation involves oxidative attack on unsaturated lipids, particularly polyunsaturated fatty acids, generating lipid radicals, lipid hydroperoxides, and reactive aldehydes such as malondialdehyde and 4-hydroxy-2-nonenal (Ayala et al., 2014; Yin et al., 2011). When lipid peroxidation occurs in mitochondrial membranes, it can alter membrane fluidity, disrupt membrane protein-lipid interactions, impair the organization of respiratory chain complexes, increase proton leak, and destabilize the protonmotive force needed for ATP synthesis (Chicco and Sparagna, 2007; Paradies et al., 2014).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:"Calibri",sans-serif">Cardiolipin is particularly important for this KER because it is a signature phospholipid of the inner mitochondrial membrane and supports the structure and function of respiratory chain complexes, supercomplexes, cytochrome c interactions, and ATP-generating membrane architecture. Oxidative modification of cardiolipin and other inner-membrane lipids can therefore reduce the efficiency with which electron transport is coupled to ATP synthesis. The downstream KE may be measured as decreased mitochondrial membrane potential, increased proton leak, reduced respiratory control ratio, lower OXPHOS coupling efficiency, or reduced ATP-generating respiratory efficiency. The KER does not require lipid peroxidation to be the only cause of decreased OXPHOS coupling, but it captures a mechanistically plausible and empirically supported route by which oxidative membrane damage can impair mitochondrial bioenergetics.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Biological plausibility of this KER is high. Lipid peroxidation can directly affect mitochondrial coupling because the inner mitochondrial membrane is both highly specialized for energy transduction and vulnerable to oxidative lipid damage. The electrochemical proton gradient that drives ATP synthesis depends on low proton conductance, intact membrane architecture, and appropriately organized electron transport chain and ATP synthase complexes. Peroxidation of phospholipids can increase membrane disorder, damage cardiolipin, alter protein-lipid interactions, facilitate proton leak, and impair respiratory chain complex function (Chicco and Sparagna, 2007; Paradies et al., 1998; Paradies et al., 2014).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The mechanistic connection is especially strong for cardiolipin. Cardiolipin stabilizes respiratory chain complexes and supercomplexes and supports cytochrome c oxidase, ATP synthase, and other components of mitochondrial bioenergetics. Cardiolipin peroxidation has been associated with loss of respiratory chain function, altered cytochrome c interactions, and mitochondrial dysfunction. Thus, increased lipid peroxidation provides a structurally and functionally credible basis for decreased coupling of OXPHOS.</span></span></span></p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Empirical support for this KER is moderate. Several studies provide concordant evidence linking lipid peroxidation or oxidative membrane damage with impaired mitochondrial membrane potential, proton leak, or OXPHOS coupling. However, fewer studies directly measure lipid peroxidation and a formal coupling metric in the same experiment across multiple time points and doses, and many available studies use related mitochondrial endpoints rather than direct OXPHOS coupling efficiency.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><strong><span style="font-family:"Calibri",sans-serif"><span style="color:black">Evidence for upstream KE 1445</span></span></strong></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><strong><span style="font-family:"Calibri",sans-serif"><span style="color:black">Evidence for downstream KE 1446</span></span></strong></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">TBARS/MDA increased significantly at >=0.5 uM paraquat.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Mitochondrial membrane potential decreased at >=0.5 uM paraquat, with dose-dependent further reduction.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Dose concordance supports association between lipid peroxidation and impaired mitochondrial polarization/coupling in the same model.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Dietary susceptibility to lipid peroxidation was associated with lower mitochondrial membrane potential, supporting a lipid damage-mitochondrial function linkage.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Supports environmental relevance of oxygen-fluctuation/oxidative damage conditions leading to reduced coupling efficiency, although lipid peroxidation itself was not the sole measured driver.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Oxidative damage to cardiolipin was observed.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Cytochrome oxidase activity was altered in close association with cardiolipin oxidative damage.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Cardiolipin loss, remodeling, and peroxidation are documented forms of mitochondrial lipid alteration.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Altered cardiolipin status is associated with mitochondrial dysfunction and reduced bioenergetic performance.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Chicco and Sparagna (2007); Paradies et al. (2014).</span></span></span></p>
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<p> </p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">A major uncertainty is that lipid peroxidation is often measured by TBARS or MDA assays, which are useful but can lack specificity and may not resolve which lipid class or subcellular membrane compartment is damaged. Because OXPHOS coupling is specifically dependent on mitochondrial inner-membrane integrity, whole-cell or whole-tissue lipid peroxidation measurements may not always provide direct information on mitochondrial lipid peroxidation. More specific measurements of cardiolipin oxidation, 4-HNE adducts, lipid hydroperoxides, or mitochondrial membrane lipidomics would strengthen evidence for this KER (Ayala et al., 2014; Yin et al., 2011).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The relationship may also be modulated by compensatory mechanisms. Mild lipid peroxidation can activate antioxidant and lipid-remodeling responses, and organisms may compensate through increased antioxidant capacity, membrane remodeling, or metabolic reorganization. Therefore, increased lipid peroxidation does not always immediately produce measurable decreases in OXPHOS coupling, especially when damage is below a threshold or when measurements are taken after compensatory recovery. Conversely, decreased OXPHOS coupling can occur through mechanisms independent of lipid peroxidation, including direct uncouplers, respiratory-chain inhibitors, protein oxidation, genetic mitochondrial defects, or ionophore-mediated proton leak.</span></span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Quantitative understanding of this KER is low to moderate. There is strong qualitative understanding that lipid peroxidation can impair mitochondrial membrane function and decrease OXPHOS coupling, but a general quantitative function linking the magnitude of lipid peroxidation to the magnitude of coupling loss is not yet established across taxa, tissues, stressors, and assay systems. The relationship is expected to be nonlinear and threshold-dependent because moderate lipid peroxidation may be buffered by antioxidants and lipid repair/remodeling, while more severe damage can abruptly increase proton leak or disrupt respiratory-chain organization.</span></span></span></p>
<strong>Response-response relationship</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">System-specific quantitative evidence exists. In Chlamydomonas reinhardtii, paraquat produced significant lipid peroxidation and decreased mitochondrial membrane potential at similar concentrations, supporting dose concordance over the tested range (Esperanza et al., 2015). In Daphnia, high-PUFA dietary conditions increased lipid peroxidation and lowered mitochondrial membrane potential, indicating a quantitative association between susceptibility to lipid oxidation and mitochondrial bioenergetic status (Moore et al., 2023). In Mya arenaria, cyclic hypoxia increased proton leak by approximately 1.5- to 1.7-fold and reduced OXPHOS coupling efficiency, supporting quantitative characterization of downstream mitochondrial uncoupling under oxidative stress-relevant conditions (Ouillon et al., 2021). However, these studies do not yet provide a single cross-system response-response equation from lipid peroxidation biomarkers to OXPHOS coupling efficiency.</span></span></span></p>
<strong>Time-scale</strong>
<p style="text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">The time scale of the linkage can range from minutes to weeks depending on the stressor and measurement strategy. Chemical peroxidation of mitochondrial lipids can affect membrane function rapidly, but whole-organism or chronic exposure studies often detect stable changes in lipid peroxidation and coupling over days to weeks. Quantitative prediction of decreased coupling from lipid peroxidation is therefore best supported in systems where mitochondrial lipid peroxidation and OXPHOS coupling are measured directly in the same cells or isolated mitochondria across a concentration and time-course series.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><strong><span style="font-family:"Calibri",sans-serif"><span style="color:black">Influence on the KER</span></span></strong></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Higher PUFA content and susceptible cardiolipin species increase vulnerability to peroxidation and may increase the probability or magnitude of decreased coupling.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Chicco and Sparagna (2007); Paradies et al. (2014); Moore et al. (2023).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Higher antioxidant capacity can reduce propagation of lipid peroxidation and buffer the effect on mitochondrial coupling; depletion increases sensitivity.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Halliwell and Gutteridge (2015); Sies et al. (2017); Ayala et al. (2014).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Can lower the threshold for lipid peroxidation and intensify mitochondrial membrane damage.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Halliwell and Gutteridge (2015); Regoli and Giuliani (2014); Knauert and Knauer (2008).</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Cyclic hypoxia or reoxygenation can increase proton leak and reduce OXPHOS coupling efficiency, potentially strengthening the KER.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Respiratory substrate, ADP availability, membrane potential and electron pressure on the ETC.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">High electron leak and high membrane potential can increase oxidative damage; pre-existing uncoupling can alter both lipid peroxidation and coupling measurements.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">TBARS, MDA, 4-HNE, lipid hydroperoxides, cardiolipin oxidation and mitochondrial lipidomics differ in specificity and time scale.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Can affect apparent dose-response and temporal concordance between the upstream and downstream KEs.</span></span></span></p>
<p><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ayala et al. (2014); Yin et al. (2011).</span></span></span></p>
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<h4>References</h4>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Almaida-Pagán PF, Lucas-Sánchez A, Tocher DR. 2014. Changes in mitochondrial membrane composition and oxidative status during rapid growth, maturation and aging in zebrafish, Danio rerio. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 1841(7):1003-1011. https://doi.org/10.1016/j.bbalip.2014.04.004.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ayala A, Munoz MF, Arguelles S. 2014. </span><span style="font-family:"Calibri",sans-serif">Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 2014:360438. https://doi.org/10.1155/2014/360438.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Chicco AJ, Sparagna GC. 2007. Role of cardiolipin alterations in mitochondrial dysfunction and disease. American Journal of Physiology-Cell Physiology 292(1):C33-C44. https://doi.org/10.1152/ajpcell.00243.2006.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Cong B, Liu C, Wang L, Chai Y. 2020. The impact on antioxidant enzyme activity and related gene expression following adult zebrafish (Danio rerio) exposure to dimethyl phthalate. Animals 10(4):717. https://doi.org/10.3390/ani10040717.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Esperanza M, Cid A, Herrero C, Rioboo C. 2015. Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: screening cytotoxicity and genotoxicity endpoints. Aquatic Toxicology 165:210-221. https://doi.org/10.1016/j.aquatox.2015.06.004.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Halliwell B, Gutteridge JMC. 2015. Free Radicals in Biology and Medicine. 5th ed. Oxford: Oxford University Press.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Knauert S, Knauer K. 2008. The role of reactive oxygen species in copper toxicity to two freshwater green algae. Journal of Phycology 44(2):311-321. https://doi.org/10.1111/j.1529-8817.2008.00471.x.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Moore TD, Martin-Creuzburg D, Yampolsky LY. 2023. Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia. Oecologia 202(1):151-163. https://doi.org/10.1007/s00442-023-05382-1.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Ouillon N, Sokolov EP, Otto S, Rehder G, Sokolova IM. 2021. Effects of variable oxygen regimes on mitochondrial bioenergetics and reactive oxygen species production in a marine bivalve Mya arenaria. Journal of Experimental Biology 224(4):jeb237156. https://doi.org/10.1242/jeb.237156.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Pan YX, Luo Z, Zhuo MQ, Wei CC, Chen GH, Song YF. 2018. Oxidative stress and mitochondrial dysfunction mediated Cd-induced hepatic lipid accumulation in zebrafish Danio rerio. </span><span style="font-family:"Calibri",sans-serif">Aquatic Toxicology 199:12-20. https://doi.org/10.1016/j.aquatox.2018.03.017.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Paradies G, Ruggiero FM, Petrosillo G, Quagliariello E. 1998. </span><span style="font-family:"Calibri",sans-serif">Peroxidative damage to cardiac mitochondria: cytochrome oxidase and cardiolipin alterations. </span><span style="font-family:"Calibri",sans-serif">FEBS Letters 424(3):155-158. https://doi.org/10.1016/S0014-5793(98)00161-6.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Paradies G, Petrosillo G, Pistolese M, Ruggiero FM. 2002. </span><span style="font-family:"Calibri",sans-serif">Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage. Gene 286(1):135-141. https://doi.org/10.1016/S0378-1119(01)00814-9.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Paradies G, Paradies V, De Benedictis V, Ruggiero FM, Petrosillo G. 2014. Functional role of cardiolipin in mitochondrial bioenergetics. </span><span style="font-family:"Calibri",sans-serif">Biochimica et Biophysica Acta - Bioenergetics 1837(4):408-417. https://doi.org/10.1016/j.bbabio.2013.10.006.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Regoli F, Giuliani ME. 2014. </span><span style="font-family:"Calibri",sans-serif">Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms. Marine Environmental Research 93:106-117. https://doi.org/10.1016/j.marenvres.2013.07.006.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Schieber M, Chandel NS. 2014. ROS function in redox signaling and oxidative stress. Current Biology 24(10):R453-R462. https://doi.org/10.1016/j.cub.2014.03.034.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Sies H, Berndt C, Jones DP. 2017. Oxidative stress. Annual Review of Biochemistry 86:715-748. https://doi.org/10.1146/annurev-biochem-061516-045037.</span></span></span></p>
<p style="margin-left:24px; text-align:justify"><span style="font-size:18px"><span style="font-family:Cambria,serif"><span style="font-family:"Calibri",sans-serif">Sokolov EP, Markert S, Hinzke T, Hirschfeld C, Becher D, Ponsuksili S, Sokolova IM. 2019. Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas. Journal of Proteomics 194:99-111.</span></span></span></p>
<td><a href="/aops/263">Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/264">Uncoupling of oxidative phosphorylation leading to growth inhibition via ATP depletion associated cell death</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/266">Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased Na-K ATPase activity</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/331">Reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/596">Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/612">Peroxisome proliferator-activated receptor alpha activation leading to early life stage mortality via reduced adenosine triphosphate</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/326">Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/332">Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/333">Reactive oxygen species leading to growth inhibition via protein oxidation and cell death</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p>Relationship 2203 is considered applicable to eukaryotes, as mitochondrial oxidative phosphorylation and ATP synthesis are highly conserved in these organisms. Uncoupling of oxidative phosphorylation leading to ATP depletion is a well-documented relationship in many taxa, such as human, rodents and fish.</p>
<p> </p>
<p><strong>Sex applicability</strong></p>
<p>Relationship 2203 is considered applicable to all genders, as mitochondrial oxidative phosphorylation and ATP synthesis are fundamental biological processes and are not sex-pecific.</p>
<p> </p>
<p><strong>Life-stage applicability</strong></p>
<p>Relationship 2203 is considered applicable to all life-stages, as mitochondrial oxidative phosphorylation and ATP synthesis are essential energy production processes for maintaining basic biological activities.</p>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify">This key event relationship describes the dissipation of protonmotive force across the inner mitochondrial membrane by uncouplers (uncoupling of oxidative phosphorylation), leading to reduced total adenosine triphosphate (ATP) pool in cells or organisms.</p>
<h4>Evidence Supporting this KER</h4>
<p style="text-align:justify"><strong>The overall evidence supporting Relationship 2203 is considered</strong> high.</p>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><strong>The biological plausibility of Relationship 2203 is considered</strong> high.</p>
<p style="text-align:justify"><strong>Rationale</strong>: In eukaryotic cells, the major metabolic pathways responsible for ATP production are OXPHOS, citric acid (TCA) cycle, glycolysis and photosynthesis. Oxidative phosphorylation is much (theoretically 15-18 times) more efficient than the rest due to high energy derived from oxygen during aerobic respiration (Schmidt-Rohr 2020). As the ATP level is relatively balanced between production and consumption (Bonora 2012), ATP depletion is a plausible consequence of reduced ATP synthetic efficiency following uncoupling of OXPHOS.</p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><strong>The empirical support of Relationship 2203 is considered</strong> high.</p>
<p><strong>Rationale:</strong> The majority of relevant studies show good incidence, temporal and/or dose concordance in different organisms and cell types after exposure to known uncouplers, with relatively few exceptions.</p>
<p><strong>Evidence</strong>:</p>
<ul>
<li><strong><em>Temporal concordance</em></strong>: Exposure of zebrafish embryos to 0.5 µM of the classical uncoupler 2,4-DNP led to significantly uncoupling of OXPHOS after 21h, whereas significant reduction in ATP was only observed after 45h <!--{C}%3C!%2D%2D%5Bendif%5D%2D%2D%2D%2D%3E-->(Bestman 2015). <!--{C}%3C!%2D%2D!%5Bendif%5D%2D%2D%2D%2D%3E--></li>
<li><strong><em>Dose concordance:</em></strong> The uncoupler triclosan induced significant uncoupling of OXPHOS in zebrafish embryos at 15 µM, whereas higher (30 µM) concentration was required to caused significant ATP depletion <!--{C}%3C!%2D%2D%5Bendif%5D%2D%2D%2D%2D%3E-->(Shim 2016).</li>
<li><!--{C}%3C!%2D%2D!%5Bendif%5D%2D%2D%2D%2D%3E--><!--{C}%3C!%2D%2D%20%2D%2D%3E--><strong><em>Dose concordance:</em></strong> Exposure to 1 µM of of the uncoupler CCCP led to 40% uncoupling of OXPHOS in rat RBL-2H3 cells, whereas the same magnitude of effect for ATP reduction required 1.6 µM of CCCP (Weatherly 2016).</li>
<li><!--{C}%3C!%2D%2D%20%2D%2D%3E--><strong><em>Dose concordance:</em></strong> Exposure to 10 µM of the uncoupler triclosan caused significant uncoupling of OXPHOS in rat RBL-2H3 cells, whereas significant reduction in ATP was observed at a higher concentration (30 µM) <!--{C}%3C!%2D%2D%5Bendif%5D%2D%2D%2D%2D%3E-->(Weatherly 2018).</li>
<li><!--{C}%3C!%2D%2D!%5Bendif%5D%2D%2D%2D%2D%3E--><!--{C}%3C!%2D%2D%20%2D%2D%3E--><strong><em>Dose concordance: </em></strong>Significant effect on uncoupling of OXPHOS required 2 µM FCCP, whereas a significant reduction in ATP required 20 µM FCCP in human RD cells <!--{C}%3C!%2D%2D%5Bendif%5D%2D%2D%2D%2D%3E-->(Kuruvilla 2003).</li>
<li><!--{C}%3C!%2D%2D!%5Bendif%5D%2D%2D%2D%2D%3E--><!--{C}%3C!%2D%2D%20%2D%2D%3E--><strong><em>Incidence concordance</em></strong>: In human colon cancer cells (SW480), exposure to 150 µM of the uncoupler flavanoid morin caused 60% reduction in MMP, whereas only around 35% decrease in ATP <!--{C}%3C!%2D%2D%5Bendif%5D%2D%2D%2D%2D%3E-->(Sithara 2017).</li>
<li><!--{C}%3C!%2D%2D!%5Bendif%5D%2D%2D%2D%2D%3E--><!--{C}%3C!%2D%2D%20%2D%2D%3E--><strong><em>Incidence concordance: </em></strong>Exposure of rat RBL-2H3 cells to 10 µM of the uncoupler triclosan led to 50% uncoupling of OXPHOS, whereas only 40% reduction in ATP (Weatherly 2016).</li>
<li><strong><em>Incidence concordance:</em></strong> Exposure to 5 µM of the uncoupler CCCP caused 71% uncoupling of OXPHOS, whereas only 64% reduction of ATP in human HL-60 cells (Sweet 1999).</li>
<li><strong><em>Incidence concordance:</em></strong> Exposure of human HeLa cells to 50 µM of the uncoupler CCCP for 1h led to 77% uncoupling of OXPHOS and 25% reduction in ATP 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<li><em><strong>Incidence concordance</strong></em>: Exposure of the nematode Caenorhabditis elegans to 50 µM Arsenite for 1h led to approximately 45% uncoupling of OXPHOS and 20% reduction in ATP (Luz 2016).</li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<ul>
<li style="text-align:justify">A significant decrease followed by a significant increase in total ATP was observed in human RD cells during a 48h exposure to the uncoupler FCCP (Kuruvilla 2003), possibly due to the enhancement of other ATP synthetic pathways (e.g., glycolysis) as a compensatory action to impaired OXPHOS (Jose 2011</li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><strong>The quantitative understanding of Relationship 2203 is</strong> high.</p>
<p style="text-align:justify"><strong>Rationale:</strong> Multiple mathematical models have been developed for describing the quantitative relationships between uncoupling of OXPHOS and ATP synthesis in vertebrates (Beard 2005; Schmitz 2011; Heiske 2017; Kubo 2020). These models, however, are highly complex metabolic or systems biological models and warrant further simplification to be used for this AOP. <!--![endif]----></p>
<strong>Response-response relationship</strong>
<p style="text-align:justify">A regression based quantitative response-response relationship between uncoupling of OXPHOS and ATP depletion was proposed for the crustacean <em>Daphnia magna</em> under UVB stress (Song 2020).</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<ul>
<li style="text-align:justify">It is known that mild uncoupling of oxidative phosphorylation can enhance the activity of the mitochondrial electron transport chain to produce more ATP, and/or activate other ATP synthetic pathways (e.g., glycolysis) as a compensatory action to impaired OXPHOS (Jose 2011).</li>
</ul>
<h4>References</h4>
<p style="text-align:justify">Beard DA. 2005. A biophysical model of the mitochondrial respiratory system and oxidative phosphorylation. PLOS Computational Biology 1:e36. DOI: 10.1371/journal.pcbi.0010036.</p>
<p>Bestman JE, Stackley KD, Rahn JJ, Williamson TJ, Chan SS. 2015. The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos. Differentiation 89:51-69. DOI: 10.1016/j.diff.2015.01.001.</p>
<p>Bonora M, Patergnani S, Rimessi A, De Marchi E, Suski JM, Bononi A, Giorgi C, Marchi S, Missiroli S, Poletti F, Wieckowski MR, Pinton P. 2012. ATP synthesis and storage. Purinergic Signalling 8:343-357. DOI: 10.1007/s11302-012-9305-8.</p>
<p>Heiske M, Letellier T, Klipp E. 2017. Comprehensive mathematical model of oxidative phosphorylation valid for physiological and pathological conditions. The FEBS Journal 284:2802-2828. DOI: <a href="https://doi.org/10.1111/febs.14151">https://doi.org/10.1111/febs.14151</a>.</p>
<p>Jose C, Bellance N, Rossignol R. 2011. Choosing between glycolysis and oxidative phosphorylation: A tumor's dilemma? Biochimica et Biophysica Acta (BBA) - Bioenergetics 1807:552-561. DOI: <a href="https://doi.org/10.1016/j.bbabio.2010.10.012">https://doi.org/10.1016/j.bbabio.2010.10.012</a>.</p>
<p>Koczor CA, Shokolenko IN, Boyd AK, Balk SP, Wilson GL, Ledoux SP. 2009. Mitochondrial DNA damage initiates a cell cycle arrest by a Chk2-associated mechanism in mammalian cells. J Biol Chem 284:36191-36201. DOI: 10.1074/jbc.M109.036020.</p>
<p>Kubo S, Niina T, Takada S. 2020. Molecular dynamics simulation of proton-transfer coupled rotations in ATP synthase FO motor. Scientific Reports 10:8225. DOI: 10.1038/s41598-020-65004-1.</p>
<p>Kuruvilla S, Qualls CW, Jr., Tyler RD, Witherspoon SM, Benavides GR, Yoon LW, Dold K, Brown RH, Sangiah S, Morgan KT. 2003. Effects of minimally toxic levels of carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone (FCCP), elucidated through differential gene expression with biochemical and morphological correlations. Toxicol Sci 73:348-361. DOI: 10.1093/toxsci/kfg084.</p>
<p>Schmidt-Rohr K. 2020. Oxygen is the high-energy molecule powering complex multicellular life: fundamental corrections to traditional bioenergetics. ACS Omega 5:2221-2233. DOI: 10.1021/acsomega.9b03352.</p>
<p>Schmitz JPJ, Vanlier J, van Riel NAW, Jeneson JAL. 2011. Computational modeling of mitochondrial energy transduction. 39:363-377. DOI: 10.1615/CritRevBiomedEng.v39.i5.20.</p>
<p>Shim J, Weatherly LM, Luc RH, Dorman MT, Neilson A, Ng R, Kim CH, Millard PJ, Gosse JA. 2016. Triclosan is a mitochondrial uncoupler in live zebrafish. J Appl Toxicol 36:1662-1667. DOI: 10.1002/jat.3311.</p>
<p>Sithara T, Arun KB, Syama HP, Reshmitha TR, Nisha P. 2017. Morin inhibits proliferation of SW480 colorectal cancer cells by inducing apoptosis mediated by reactive oxygen species formation and uncoupling of Warburg effect. Frontiers in Pharmacology 8. DOI: 10.3389/fphar.2017.00640.</p>
<p>Song Y, Xie L, Lee Y, Tollefsen KE. 2020. De novo development of a quantitative adverse outcome pathway (qAOP) network for ultraviolet B (UVB) radiation using targeted laboratory tests and automated data mining. Environmental Science & Technology 54:13147-13156. DOI: 10.1021/acs.est.0c03794.</p>
<p>Sweet S, Singh G. 1999. Changes in mitochondrial mass, membrane potential, and cellular adenosine triphosphate content during the cell cycle of human leukemic (HL-60) cells. Journal of Cellular Physiology 180:91-96. DOI: <a href="https://doi.org/10.1002/(SICI)1097-4652(199907)180:1">https://doi.org/10.1002/(SICI)1097-4652(199907)180:1</a><91::AID-JCP10>3.0.CO;2-6.</p>
<p>Weatherly LM, Nelson AJ, Shim J, Riitano AM, Gerson ED, Hart AJ, de Juan-Sanz J, Ryan TA, Sher R, Hess ST, Gosse JA. 2018. Antimicrobial agent triclosan disrupts mitochondrial structure, revealed by super-resolution microscopy, and inhibits mast cell signaling via calcium modulation. Toxicol Appl Pharmacol 349:39-54. DOI: 10.1016/j.taap.2018.04.005.</p>
<p>Weatherly LM, Shim J, Hashmi HN, Kennedy RH, Hess ST, Gosse JA. 2016. Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes. Journal of Applied Toxicology 36:777-789. DOI: <a href="https://doi.org/10.1002/jat.3209">https://doi.org/10.1002/jat.3209</a>.</p>
</div>
<div>
<h4><a href="/relationships/2204">Relationship: 2204: Decrease, ATP pool leads to Decrease, Cell proliferation</a></h4>
<td><a href="/aops/263">Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/290">Mitochondrial ATP synthase antagonism leading to growth inhibition (1)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/286">Mitochondrial complex III antagonism leading to growth inhibition (1)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/601">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/326">Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/332">Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p>Relationship 2204 is considered applicable to all eukaryotes, as ATP and cell proliferation are known to be tightly coupled in animals, plants and some microorganisms.</p>
<p> </p>
<p><strong>Sex applicability</strong></p>
<p>Relationship 2204 is considered applicable to all sexes, as ATP-dependent cell proliferation are used by both males and females in eukaryotes.</p>
<p> </p>
<p><strong>Life-stage applicability</strong></p>
<p>Relationship 2204 is considered applicable to all life stages, as ATP-dependent cell proliferation is an essential process for an organism throughout the entire life.</p>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify">This key event relationship describes reduced adenosine triphosphate (ATP) supply leading to reduced cell proliferation (cell growth, division or a combination of these).</p>
<h4>Evidence Supporting this KER</h4>
<p style="text-align:justify"><strong>The overall evidence supporting Relationship 2204 is considered</strong> moderate.</p>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><strong>The biological plausibility of Relationship 2204 is considered</strong> high.</p>
<p><strong>Rationale</strong>: Cell proliferation is a well-known ATP-dependent process. Cell division processes, such as the mitotic cell cycle uses ATP for chromosome movements and DNA replication (Kingston 1999). The synthetic processes of major cellular components that are necessary for cell structure and growth, such as proteins and lipids, also require sufficient ATP supply (Bonora 2012). Depletion of ATP therefore has a negative impact on these processes.</p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><strong>The empirical support of Relationship 2204 is considered</strong> moderate.</p>
<p><strong>Evidence</strong>:</p>
<ul>
<li><em><strong>Incidence concordance</strong></em>: Exposure of human HeLa cells to 50 µM of the uncoupler CCCP for 1h led to 25% reduction in ATP, whereas a non-significant reduction in cell proliferation (Koczor 2009).</li>
<li>I<em><strong>ncidence concordance</strong></em>: Exposure of human RD cells to 20 µM of the uncoupler CCCP for 2h led to 20% ATP depletion, whereas a non-significant decrease in cell proliferation (Kuruvilla 2003).</li>
<li><em><strong>Incidence concordance</strong></em>: Exposure of human SE480 cells to 150 µM of the uncoupler flavanoid morin for 48h led to 35% ATP depletion and 35% reduction in cell proliferation (Sithara 2017).</li>
<strong>Uncertainties and Inconsistencies</strong>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:等线"><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">There are currently no inconsistencies based on the supporting literature.</span></span></span></span></p>
<p style="text-align:justify">Ahmann FR, Garewal HS, Schifman R, Celniker A, Rodney S. 1987. Intracellular adenosine triphosphate as a measure of human tumor cell viability and drug modulated growth. In Vitro Cellular & Developmental Biology 23:474-480. DOI: 10.1007/BF02628417.</p>
<p>Bonora M, Patergnani S, Rimessi A, De Marchi E, Suski JM, Bononi A, Giorgi C, Marchi S, Missiroli S, Poletti F, Wieckowski MR, Pinton P. 2012. ATP synthesis and storage. Purinergic Signalling 8:343-357. DOI: 10.1007/s11302-012-9305-8.</p>
<p>Crouch SPM, Kozlowski R, Slater KJ, Fletcher J. 1993. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. Journal of Immunological Methods 160:81-88. DOI: <a href="https://doi.org/10.1016/0022-1759(93)90011-U">https://doi.org/10.1016/0022-1759(93)90011-U</a>.</p>
<p>Kingston RE, Narlikar GJ. 1999. ATP-dependent remodeling and acetylation as regulators of chromatin fluidity. Genes Dev 13:2339-2352. DOI: 10.1101/gad.13.18.2339.</p>
<p>Koczor CA, Shokolenko IN, Boyd AK, Balk SP, Wilson GL, Ledoux SP. 2009. Mitochondrial DNA damage initiates a cell cycle arrest by a Chk2-associated mechanism in mammalian cells. J Biol Chem 284:36191-36201. DOI: 10.1074/jbc.M109.036020.</p>
<p>Kuruvilla S, Qualls CW, Jr., Tyler RD, Witherspoon SM, Benavides GR, Yoon LW, Dold K, Brown RH, Sangiah S, Morgan KT. 2003. Effects of minimally toxic levels of carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone (FCCP), elucidated through differential gene expression with biochemical and morphological correlations. Toxicol Sci 73:348-361. DOI: 10.1093/toxsci/kfg084.</p>
<p>Nieminen AL, Saylor AK, Herman B, Lemasters JJ. 1994. ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition. Am J Physiol 267:C67-74. DOI: 10.1152/ajpcell.1994.267.1.C67.</p>
<p>Sithara T, Arun KB, Syama HP, Reshmitha TR, Nisha P. 2017. Morin inhibits proliferation of SW480 colorectal cancer cells by inducing apoptosis mediated by reactive oxygen species formation and uncoupling of Warburg effect. Frontiers in Pharmacology 8. DOI: 10.3389/fphar.2017.00640.</p>
<td><a href="/aops/263">Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/290">Mitochondrial ATP synthase antagonism leading to growth inhibition (1)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/286">Mitochondrial complex III antagonism leading to growth inhibition (1)</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/267">Uncoupling of oxidative phosphorylation leading to growth inhibition via glucose depletion</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/602">Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/603">Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/601">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/324">Reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell cycle disruption</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/326">Reactive oxygen species leading to growth inhibition via lipid peroxidation and decreased cell proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/332">Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p>Relationship 2205 is considered applicable to all eukaryotes (both unicellular and multicellular), as growth (or population growth of alga) is well known to be achieved through cell proliferation in animals, plants and some microorganisms.</p>
<p>Relationship 2205 is considered applicable to both all sexes, as cell proliferation leading to growth is a fundamental process and not sex-specific.</p>
<p>Relationship 2205 is considered applicable to all life stages, as cell proliferation leading to growth is essential for maintaining basic biological processes throughout an organism’s life.</p>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify">This key event relationship describes reduced cell proliferation (cell growth, division or a combination of these) leading to reduced tissue, organ or individual growth.</p>
<h4>Evidence Supporting this KER</h4>
<p style="text-align:justify"><strong>The overall evidence supporting Relationship 2205 is considered</strong> moderate.</p>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><strong>The biological plausibility of Relationship 2205 is considered</strong> high.</p>
<p><strong>Rationale</strong>: The biological structural and functional relationship between cell proliferation and growth is well established. It is commonly accepted that the size of an organism, organ or tissue is dependent on the total number and volume of the cells it contains, and the amount of extracellular matrix and fluids (Conlon 1999). Impairment to cell proliferation can logically affect tissue and organismal growth.</p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><strong>The empirical support of Relationship 2205 is considered</strong> low.</p>
<p><strong>Rationale</strong>: Because cell proliferation is typically measured in vitro, while growth of an organism is measured in vivo, few studies have measured both in the same experiment. There is one zebrafish study reporting concordant relationship between reduced cell proliferation and embryo growth with some inconsistencies (Bestman 2015). <!--![endif]----></p>
<strong>Uncertainties and Inconsistencies</strong>
<ul>
<li style="text-align:justify">In zebrafish embryos exposed to 2,4-DNP, significant growth inhibition (AO), as indicated by whole embryo length, caudal primary (CaP) motor neuron axons and otic vesicle length (OVL) ratio after 21h, somite width and eye diameter after 45h exposure was identified, after 21h, whereas a non- significant reduction in cell proliferation was observed (Bestman 2015).</li>
<p style="text-align:justify">Bestman JE, Stackley KD, Rahn JJ, Williamson TJ, Chan SS. 2015. The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos. Differentiation 89:51-69. DOI: 10.1016/j.diff.2015.01.001.</p>
<p>Binder BJ, Landman KA, Simpson MJ, Mariani M, Newgreen DF. 2008. Modeling proliferative tissue growth: a general approach and an avian case study. Phys Rev E Stat Nonlin Soft Matter Phys 78:031912. DOI: 10.1103/PhysRevE.78.031912.</p>
<p>Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.</p>
<p>Jarrett AM, Lima EABF, Hormuth DA, McKenna MT, Feng X, Ekrut DA, Resende ACM, Brock A, Yankeelov TE. 2018. Mathematical models of tumor cell proliferation: A review of the literature. Expert Review of Anticancer Therapy 18:1271-1286. DOI: 10.1080/14737140.2018.1527689.</p>
<p>Mosca G, Adibi, M., Strauss, S., Runions, A., Sapala, A., Smith, R.S. 2018. Modeling Plant Tissue Growth and Cell Division. In Morris R., ed, Mathematical Modelling in Plant Biology. Springer, Cham.</p>