<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/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell death</a></td>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation</a></td>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/327">Aop:327 - Excessive reactive oxygen species production leading to mortality (1)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/328">Aop:328 - Excessive reactive oxygen species production leading to mortality (2)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/329">Aop:329 - Excessive reactive oxygen species production leading to mortality (3)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/330">Aop:330 - Excessive reactive oxygen species production leading to mortality (4)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/26">Aop:26 - Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/534">Aop:534 - Succinate dehydrogenase (SDH) inhibition leads to cancer through oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/273">Aop:273 - Mitochondrial complex inhibition leading to liver injury</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/488">Aop:488 - Increased reactive oxygen species production leading to decreased cognitive function</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/298">Aop:298 - Increase in reactive oxygen species (ROS) leading to human treatment-resistant gastric cancer via chronic ROS</a></td>
<td><a href="/aops/298">Aop:298 - Increase in reactive oxygen species (ROS) leading to human treatment-resistant gastric cancer</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/27">Aop:27 - Cholestatic Liver Injury induced by Inhibition of the Bile Salt Export Pump (ABCB11)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/511">Aop:511 - The AOP framework on ROS-mediated oxidative stress induced vascular disrupting effects </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/207">Aop:207 - NADPH oxidase and P38 MAPK activation leading to reproductive failure in Caenorhabditis elegans</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/423">Aop:423 - Toxicological mechanisms of hepatocyte apoptosis through the PARP1 dependent cell death pathway </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/481">Aop:481 - AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/282">Aop:282 - Adverse outcome pathway on photochemical toxicity initiated by light exposure</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/569">Aop:569 - Decreased DNA methylation of FAM50B/PTCHD3 leading to IQ loss of children via PI3K-Akt pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<p>ROS is a normal constituent found in all organisms, <em>lifestages, and sexes.</em></p>
<h4>Key Event Description</h4>
<p>Biological State: increased reactive oxygen species (ROS)</p>
<p>Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.</p>
<p>Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
<p>Reactive oxygen species (ROS) are O<sub>2</sub>- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). </p>
<div>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Reactive oxygen species (ROS) refers to the chemical species superoxide, hydrogen peroxide, and their secondary reactive products. In the biological context, ROS are signaling molecules with important roles in cell energy metabolism, cell proliferation, and fate. Therefore, balancing ROS levels at the cellular and tissue level is an important part of many biological processes. Disbalance, mainly an increase in ROS levels, can cause cell dysfunction and irreversible cell damage.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are produced from both exogenous stressors and normal endogenous cellular processes, such as the mitochondrial electron transport chain (ETC). Inhibition of the ETC can result in the accumulation of ROS. Exposure to chemicals, heavy metal ions, or ionizing radiation can also result in increased production of ROS. Chemicals and heavy metal ions can deplete cellular antioxidants reducing the cell’s ability to control cellular ROS and resulting in the accumulation of ROS. Cellular antioxidants include glutathione (GSH), protein sulfhydryl groups, superoxide dismutase (SOD). </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are radicals, ions, or molecules that have a single unpaired electron in their outermost shell of electrons, which can be categorized into two groups: free oxygen radicals and non-radical ROS [Liou et al., 2010]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">highly reactive lipid- or carbohydrate-derived carbonyl compounds</span></span></p>
</td>
</tr>
</tbody>
</table>
</div>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Potential sources of ROS include NADPH oxidase, xanthine oxidase, mitochondria, nitric oxide synthase, cytochrome P450, lipoxygenase/cyclooxygenase, and monoamine oxidase [Granger et al., 2015]. ROS are generated through NADPH oxidases consisting of p47<sup>phox</sup> and p67<sup>phox</sup>. ROS are generated through xanthine oxidase activation in sepsis [Ramos et al., 2018]. Arsenic produces ROS [Zhang et al., 2011]. Mitochondria-targeted paraquat and metformin mediate ROS production [Chowdhury et al., 2020]. ROS are generated by bleomycin [Lu et al., 2010]. Radiation induces dose-dependent ROS production [Ji et al., 2019]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are generated in the course of cellular respiration, metabolism, cell signaling, and inflammation [Dickinson and Chang 2011; Egea et al. 2017]. Hydrogen peroxide is also made by the endoplasmic reticulum in the course of protein folding. Nitric oxide (NO) is produced at the highest levels by nitric oxide synthase in endothelial cells and phagocytes. NO production is one of the main mechanisms by which phagocytes kill bacteria [Wang et al., 2017]. The other species are produced by reactions with superoxide or peroxide, or by other free radicals or enzymes.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS activity is principally local. Most ROS have short half-lives, ranging from nano- to milliseconds, so diffusion is limited, while reactive nitrogen species (RNS) nitric oxide or peroxynitrite can survive long enough to diffuse across membranes [Calcerrada et al. 2011]. Consequently, local concentrations of ROS are much higher than average cellular concentrations, and signaling is typically controlled by colocalization with redox buffers [Dickinson and Chang 2011; Egea et al. 2017]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Although their existence is limited temporally and spatially, ROS interact with other ROS or with other nearby molecules to produce more ROS and participate in a feedback loop to amplify the ROS signal, which can increase RNS. Both ROS and RNS also move into neighboring cells, and ROS can increase intracellular ROS signaling in neighboring cells [Egea et al. 2017].</span></span></p>
<p>In the primary event, photoreactive chemicals are excited by the absorption of photon energy. The energy of the photoactivated chemicals transfer to oxygen and then generates the reactive oxygen species (ROS), including superoxide (O<sub>2</sub><sup>−</sup>) via type I reaction and singlet oxygen (<sup>1</sup>O<sub>2</sub>) via type II reaction, as principal intermediate species in phototoxic reaction (Foote, 1991, Onoue et al. , 2009).</p>
</div>
<h4>How it is Measured or Detected</h4>
<p>Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies.</p>
<p>Yuan, Yan, et al., (2013) described ROS monitoring by using H<sub>2</sub>-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H<sub>2</sub>-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm.</p>
<p>Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013).</p>
<p>Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS.</p>
<p>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/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/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<p dir="ltr">ROS is a normal constituent found in all organisms, therefore, all organisms containing lipid membranes may be affected by lipid peroxidation. </p>
<p>Structure: Regardless of sex or life stage, when exposed to free radicals, there is potential for lipid peroxidation as a auxiliary response where there are lipid membranes.</p>
<h4>Key Event Description</h4>
<p>Lipid peroxidation is the direct damage to lipids in the membrane of the cell or the membranes of the organelles inside the cells. Ultimately the membranes will break due to the build-up damage in the lipids. This is mainly caused by oxidants which attack lipids specifically, since these contain carbon-carbon double bonds. During lipid peroxidation several lipid radicals are formed in a chain reaction. These reactions can interfere and stimulate each other. Antioxidants, such as vitamin E, can react with lipid peroxy radicals to prevent further damage in the cell (Cooley et al. 2000).</p>
<h4>How it is Measured or Detected</h4>
<p>The main product of lipid peroxidation, malondialdehyde and 4-hydroxyalkenals, is used to measure the degree of this process. This is measured by photocolorimetric assays, quantification of fatty acids by gaseous liquid chromatography (GLC) or high performance (HPLC) (L. Li et al. 2019; Jin et al. 2010a) or through commercial kits purchased from specialized companies.</p>
<p> </p>
<h4>References</h4>
<p>Cooley HM, Evans RE, Klaverkamp JF. 2000. Toxicology of dietary uranium in lake whitefish (Coregonus clupeaformis). Aquatic Toxicology. 48(4):495–515. https://doi.org/10.1016/S0166-445X(99)00057-0</p>
<p>Jin, Yuanxiang, Xiangxiang Zhang, Linjun Shu, Lifang Chen, Liwei Sun, Haifeng Qian, Weiping Liu, and Zhengwei Fu. 2010a. “Oxidative Stress Response and Gene Expression with Atrazine Exposure in Adult Female Zebrafish (Danio Rerio).” Chemosphere 78 (7): 846–52.</p>
<p>Li, Luxiao, Shanshan Zhong, Xia Shen, Qiujing Li, Wenxin Xu, Yongzhen Tao, and Huiyong Yin. 2019. “Recent Development on Liquid Chromatography-Mass Spectrometry Analysis of Oxidized Lipids.” Free Radical Biology & Medicine 144 (November): 16–34.</p>
<td><a href="/aops/212">Aop:212 - Histone deacetylase inhibition leading to testicular atrophy</a></td>
<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/393">Aop:393 - AOP for thyroid disorder caused by triphenyl phosphate via TRβ activation</a></td>
<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/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and reduced cell proliferation</a></td>
<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/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/260">Aop:260 - CYP2E1 activation and formation of protein adducts leading to neurodegeneration</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/450">Aop:450 - Inhibition of AChE and activation of CYP2E1 leading to sensory axonal peripheral neuropathy and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/501">Aop:501 - Excessive iron accumulation leading to neurological disorders</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/471">Aop:471 - Various neuronal effects induced by elavl3, sox10, and mbp</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/31">Aop:31 - Oxidation of iron in hemoglobin leading to hematotoxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/534">Aop:534 - Succinate dehydrogenase (SDH) inhibition leads to cancer through oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/462">Aop:462 - Activation of reactive oxygen species leading the atherosclerosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<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>
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<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>
<h4><a href="/events/1446">Event: 1446: Decrease, Coupling of oxidative phosphorylation</a></h4>
<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>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/534">Aop:534 - Succinate dehydrogenase (SDH) inhibition leads to cancer through oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/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>
<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>The disruption of the cell cycle leads to a decrease in cell number. The cell cycle consists of G<sub>1</sub>, S, G<sub>2</sub>, M, and G<sub>0</sub> phases. The cell cycle regulation is disrupted by the cell cycle arrest in certain cell cycle phases. The histone gene expression is regulated in cell cycle phases [Heintz et al., 1983].</p>
<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>The percentage of cells at G<sub>1</sub>, G<sub>0</sub>, S, and G<sub>2</sub>/M phases can be detected by flow cytometry [Li et al., 2013]. Cell cycle distribution was analyzed by fluorescence-activated cell sorter (FACS) analysis with a Partec PAS-II sorter [Zupkovitz et al., 2010]. The four cell-cycle phases in living cells can be measured with four-color fluorescent proteins using live-cell imaging [Bajar et al., 2016]. The incorporation of [<sup>3</sup>H]deoxycytidine or [<sup>3</sup>H]thymidine into cell DNA during the S phase can be monitored as DNA synthesis [Heintz et al., 1982].</p>
<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>
</ul>
<h4>References</h4>
<p>Bajar, B.T. et al. (2016), "Fluorescent indicators for simultaneous reporting of all four cell cycle phases", Nat Methods 13:993-996 </p>
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>Heintz, N. et al. (1983), "Regulation of human histone gene expression: Kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle", Molecular and Cellular Biology 3:539-550</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>Li, Q. et al. (2013), "Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis", Drug Des Devel Ther 7:635-643</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>
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<td><a href="/aops/329">Aop:329 - Excessive reactive oxygen species production leading to mortality (3)</a></td>
<td>KeyEvent</td>
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<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>
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<td><a href="/aops/263">Aop:263 - Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/290">Aop:290 - Mitochondrial ATP synthase antagonism leading to growth inhibition (1)</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/291">Aop:291 - Mitochondrial ATP synthase antagonism leading to growth inhibition (2)</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/286">Aop:286 - Mitochondrial complex III antagonism leading to growth inhibition (1)</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/399">Aop:399 - Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td><a href="/aops/287">Aop:287 - Mitochondrial complex III antagonism leading to growth inhibition (2)</a></td>
<td>KeyEvent</td>
</tr>
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<td><a href="/aops/460">Aop:460 - Antagonism of Smoothened receptor leading to orofacial clefting</a></td>
<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>
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<td><a href="/aops/267">Aop:267 - Uncoupling of oxidative phosphorylation leading to growth inhibition via glucose depletion</a></td>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
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<td><a href="/aops/502">Aop:502 - Decrease, cholesterol synthesis leads to orofacial clefting</a></td>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
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<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and reduced cell proliferation</a></td>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation</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>
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<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>
<p>This key event is in general applicable to all eukaryotes, as most organisms are known to use cell proliferation to achieve growth.</p>
<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>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 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>This key event is sex-unspecific, as both genders use the same cell proliferation mechanisms.</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 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>
<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">Multiple types of <em>in vitro</em> bioassays can be used to measure this key event:</p>
<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>
<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>
<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>
<p style="text-align:justify"> </p>
<p><!--![endif]----></p>
<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>
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 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>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 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>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>
<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/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
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<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><a href="/aops/263">Aop:263 - Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation</a></td>
<td>AdverseOutcome</td>
</tr>
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<td><a href="/aops/290">Aop:290 - Mitochondrial ATP synthase antagonism leading to growth inhibition (1)</a></td>
<td>AdverseOutcome</td>
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<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/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and cell death</a></td>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell death</a></td>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and reduced cell proliferation</a></td>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation</a></td>
<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>
<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/413">Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure</a></td>
<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/492">Glutathione conjugation leading to reproductive dysfunction via oxidative stress</a></td>
<td><a href="/aops/26">Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/534">Succinate dehydrogenase (SDH) inhibition leads to cancer through oxidative stress</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/521">Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td><a href="/aops/481">AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/324">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/325">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/326">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/331">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/332">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/333">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/596">Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/599">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/325">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<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/332">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<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/329">Excessive reactive oxygen species production leading to mortality (3)</a></td>
<td><a href="/aops/602">Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/603">Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p>Considering the empirical domain of the evidence, the increased, reactive oxygen species leading to increased, lipid peroxidation is known to occur in fish and mammals, but, based on scientific reasoning, the biologically plausible domain of applicability can be eukaryotic organisms in general. It can be measured at any stage of life and in both male and female species.</p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Life Stage: The life stage applicable to this key event relationship is all life stages. Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles > embryos) due to accumulation of reactive oxygen species.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sex: This key event relationship applies to both males and females.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Taxonomic: This key event relationship appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats), teleost fish, and invertebrates (cladocerans, mussels).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p>Induction of oxidative stress occurs as a result of an imbalance between the production of radical species and the antioxidant defense systems (Juan et al. 2021). ROS can damage DNA, lipids, and proteins (Shields et al. 2021). Superoxide dismutase is an enzyme in a common cellular defense pathway, in which superoxide dismutase converts superoxide radicals to hydrogen peroxide. When cellular defense mechanisms are unable to mitigate ROS formation from mitochondrial respiration and stressors (biological, chemical, radiation), increased ROS levels cause oxidative stress.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Biological plausibility of this KER lies in the fact that reactive species, in excess, react and change macromolecules such as proteins, nucleic acids and lipids. Membrane lipids are particularly susceptible to damage by free radicals, as they are composed by unsaturated fatty acids (Su et al. 2019). Hence, increase in ROS production beyond antioxidant system defense capability of cells enables free circulation of molecules such as O2·−, HO·, H2O2, which removes electrons from membrane lipids and then triggers lipid peroxidation (Auten and Davis 2009; Su et al. 2019). </span></p>
<p>The biological plausibility linking increases in oxidative stress to reactive oxygen species (ROS) is strong. Reactive oxygen species (ROS) are produced by many normal cellular processes (ex. cellular respiration, mitochondrial electron transport, specialized enzyme reactions) and occur in multiple chemical forms (ex. superoxide anion, hydroxyl radical, hydrogen peroxide). Antioxidant enzymes play a major role in reducing reactive oxygen species (ROS) levels in cells (Ray et al. 2012) to prevent cellular damage to lipids, proteins, and DNA (Juan et al. 2021). Oxidative stress occurs when antioxidant enzymes do not prevent ROS levels from increasing in cells, often induced by environmental stressors (biological, chemical, radiation).</p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Analyses performed to support this relation show that KER3 is unchained by the three previously selected xenobiotics, as well as it takes place in a conserved way among species. Connection among the KEs is observed in both in vitro experimental models and in vivo systems, including fishes, birds and mammals.</span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">In cultures of rat hepatocytes, progressive ROS increase during 4 hours of treatment, triggered by DEM (5 mM), is followed by a continuous growth in levels of thiobarbituric acid reactive substances (TBARS), lipid peroxidation markers (Tirmenstein et al. 2000). This chemical depletes GSH content, leading to an augmentation of ROS levels and, consequently, to lipid peroxidation. In an in vivo model, 52 μM of DEM intraperitoneally injected in male Balb/c mice for two weeks caused a significant decrease in the GSH, increase in GSSG, ROS generation and increase in lipid peroxidation in testicles (Kalia and Bansal 2008).</span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">ATZ (46.4 µM) causes an increase of 48.97% of ROS and of 12.5% in MDA content in cultures of Sertoli-Germ cells from Wistar rats (25–28 days old), after, respectively, 3 and 24 h post-exposure. At a higher concentration (232 µM), these cells reach a maximum peak of ROS production after 6h of exposure, while MDA generation gets to the peak only after 24 h of treatment (Abarikwu, Pant, and Farombi 2012). In in vivo model, ATZ (38.5, 77 e 154 mg/Kg bw/day) led to a decrease in total antioxidant capacity (TAC) in a dose-dependent manner in male Sprague-Dawley rats of Specific Pathogen Free (SPF) ATZ-treated for 30 days. Which indirectly suggests increase in ROS levels – and increased malondialdehyde (MDA) content in 154 mg/Kg (Song et al. 2014). </span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">In relation to Hg, it was found that male young Wistar rats exposed to an initial dose of 4.6 μg/Kg of this metal (with following doses of 0.07 μg/Kg/day) displayed an increase in ROS levels, followed by an elevation of MDA content in testicles and epididymis of these rats 60 days post-exposure (Rizzetti et al. 2017). Other assays still carried out with male rats showed that the heavy metal induces oxidative stress with a single subcutaneous dose of 5 mg/Kg, by a substantial diminishment of activity of the main testicle antioxidant enzymes: SOD, CAT and GPX. Consequently, blood hydroperoxide and testicle MDA levels rose in a relevant way (El-Desoky et al. 2013).</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Furthermore, Hy-Line Brown laying hens fed with 4 experimental diets containing graded levels of Hg at 0.280, 3.325, 9.415, and 27.240 mg/Kg, respectively, for 10 weeks had GSH content significantly decreased in all Hg-treatment groups in ovaries, whilst SOD, CAT, GPX and glutathione reductase (GR) enzyme activities were significantly reduced, pointing to ROS accumulation. MDA content strongly increased in the 27.240-mg/Kg Hg group (Ma et al. 2018). </span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Hence, it can be deduced that, as in other adjacent relations evaluated, there is also evidence here that upstream KE is initially required in order to downstream KE take place, which reaffirms time concordance. Besides this, data enhance dose and incidence concordances for this KER.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Mechanisms involving lipid peroxidation, such as that one caused by ROS accumulation in cells, have been investigated for decades (Tirmenstein et al. 2000; Yin, Xu, and Porter 2011; Su et al. 2019). For this reason, there is much experimental data about response-response relationships or a growth of upstream KE in relation to downstream KE.</span></p>
<strong>Response-response relationship</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">This mechanism can be better understood through a process chain that consists of initiation, propagation and termination, as discussed by (Yin, Xu, and Porter 2011). In their review, these authors summarized a series of chemical reactions that develop during all this self-oxidation process and represent them in a schematic manner, as displayed in figure below.</span></p>
<p style="text-align:center"><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Lu et al. 2016; Alomar et al. 2017; Chen et al. 2017; Veneman et al. 2017; Barboza et al. 2018; Choi et al. 2018; Espinosa et al. 2018</span></span></span></p>
<p style="text-align:center"><span style="font-size:16px"><span style="font-family:Calibri,sans-serif"><span style="color:black">Browne et al. 2013; Jeong et al. 2016, 2017; Paul-Pont et al. 2016; Lei et al. 2018; Yu et al. 2018</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Furthermore, although phospholipid oxidizability is lower, once their rate of diffusion in membranes is slower, the kinetics for this kind of reaction shown in figure follows the same law of velocity (steady-state rate) of homogeneous systems (equation below) (Yin, Xu, and Porter 2011). Oxygen consumption of the equation represents the rate of steady state, while rate of radical generation is defined by R<sub>i</sub>, the constant of propagation rate is expressed as k<sub>p</sub> and the termination rate constant for the reaction is called k<sub>t</sub>.</span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">The accumulation of reactive oxygen species (ROS), and resulting oxidative stress, is well-established (see Shields 2021 for overview). In the studies listed in the above table, changes in enzyme activity and changes in gene expression are the most common oxidative stress effects detected due to increases in reactive oxygen species (see additional study details in table below). Increases in gene expression or enzyme activity of superoxide dismutase, catalase, glutathione peroxidase, and other antioxidants are frequently used as indicators of oxidative stress.</span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">For instance, empirical evidences show that rat hepatocytes begin ROS production after the first 30 minutes of DEM exposition (5 mM), growing linearly for all the remaining time, whereas the increase in products of lipid peroxidation (TBARS) starts only from the first hour of exposure (Tirmenstein et al. 2000).</span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Diet exposure of 0.01, 0.1, 0.5 mg/day of 5 and 20 um polystyrene microplastic particles.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Five-week old male mice showed changes in enzyme levels responsible for eliminating ROS. Decreased catalase at 0.1/0.5 mg/day, increased glutathione peroxidase at all doses, increased superoxide dismutase at all doses.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cerebral and epithelial human cell lines showed measured increased percent effect of ROS (as superoxide generated) with corresponding decreases in cell viability.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 20, 200, 2000 ug/L of 5 and 20 um polystyrene microplastics.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult five-month old fish showed changes in enzyme levels responsible for eliminating ROS. Increased catalase at 200/2000 ug/L, increased superoxide dismutase at all doses.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Survey of wild fish with microplastic ingestion versus no microplastic ingestion.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 0.010, 0.016 mg/L of Mercury chloride, 0.26, 0.69 mg/L of 1-5 um polymer microspheres, and mixture.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Juvenile fish showed increased ROS (Brain and muscle lipid peroxidation levels) and corresponding changes in enzyme levels (increases in muscle lactate dehydrogenase, decreases in isocitrate dehydrogenase). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 50, 250 mg/L of 150-180 um, 300-355 um polyethylene microspheres</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In vitro exposure of 100 mg/L of polyvinylchloride and polyethylene microplastics</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fish head-kidney leucocytes showed increased gene expression of nuclear factor (nrf2), associated with oxidative stress, only statistically significant in S. aurata.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Lugworms showed decreased ability to respond to ROS by ferric reducing antioxidant power (FRAP) assay, statistically significant only with phenanthrene.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 10 ug/mL of 0.05, 0.5, 6 um diameter polystyrene microbeads.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rotifers showed increased ROS levels, changes in phosphorylation of MAPK signaling proteins, and corresponding changes in enzyme and protein levels (decreased glutathione, increased superoxide dismutase, increased glutathione reductase, increased glutathione reductase, glutathione S-transferase). Enzyme statistical significance was seen most frequently with 0.05 diameter size class).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 20 ug/mL of 0.05, 0.5, 6 um diameter polystyrene microbeads.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Copepods showed increased ROS for 0.05 um diameter size class only. Corresponding increases in enzymes were also seen only in 0.05 um diameter size class (glutathione reductase, glutathione peroxidase, glutathione S-transferase, superoxide disumutase).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aquatic exposure of 30 ug/L fluoranthene, 32 ug/L of 2 and 6 um polystyrene microbeads, and mixture for 7 days and depuration for 7 days.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mussels showed increased ROS production in all treatments for 7 days, changes in enzyme and gene levels were observed for catalase, superoxide dismutase, glutathione S-transferase, glutathione reductase, and lipid peroxidation, statistical significance was not always observed.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yu et al. (2018)</span></span></p>
</td>
</tr>
</tbody>
</table>
</div>
<p>1 Assumed: study selected stressor(s) known to elevate reactive oxygen species (ROS) levels, endpoints verified increased oxidative stress and disrupted pathway.</p>
<p> </p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>The reactive oxygen species (ROS) increase needed to elicit oxidative stress is highly dependent on many other variables including age, tissue, sex, nutritional status, and co-exposures to other stressors. It is consistently characterised as an 'excess' of ROS in order to create a state of oxidative stress. Consequently, the quantitative relationship is not easily generalized. </p>
<p><!--EndFragment --></p>
<p>Some examples of normal levels have been reported at 1-8 μM (H2O2) in normal human plasma, while only 1 μM ROS present in healthy cells (Lacy et al. 2000). Inflammatory lung diseases can cause H2O2 excesses to the level of a 20-fold increment. It can also cause the level of H2O2 in ischemia and reperfusion to reach 160 μM (Burgoyne et al. 2013).</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>AP-1 and NF-κB are ROS sensing transcription factors and act as redox sensors due to the presence of a single Cys in their DNA-binding domains (Abate et al. 2006). Oxidation of these Cys residues blocks their binding to the respective consensus DNA sequences. Apurinic/apyrimidinic (AP) endonuclease 1 (APE1), functions as a reducing agent for various transcription factors (Evans et al. 2000). This ubiquitous multifunctional protein is induced by ROS (Ramana et al. 1998) and is involved in base excision repair (Demple and Sung 2005). Although reducing condition is favorable for DNA binding, both AP-1 and NF-κB can be activated by oxidative stress via induction of APE1. A Zn-finger DNA-binding protein, early growth response gene-1 (Egr-1), is activated by ROS, and a positive feedback loop between APE1 and Egr-1 regulates their early transcriptional activation after oxidative stress (Pines et al. 2005). Egr-1 also induces SOD1 and thus reduces free radical-induced damage (Minc et al. 1999).</p>
<h4>References</h4>
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<p>Abate, C., Patel, L., Rauscher III, F. J., & Curran, T. (1990). Redox regulation of fos and jun DNA-binding activity in vitro. Science, 249(4973), 1157-1161.</p>
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<p>Burgoyne, J. R., Oka, S. I., Ale-Agha, N., & Eaton, P. (2013). Hydrogen peroxide sensing and signaling by protein kinases in the cardiovascular system. Antioxidants & redox signaling, 18(9), 1042-1052.</p>
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<p>Demple, B., & Sung, J. S. (2005). Molecular and biological roles of Ape1 protein in mammalian base excision repair. DNA repair, 4(12), 1442-1449.</p>
<p>Deng, Y., Zhang, Y., Lemos, B., and Ren, H. 2017. Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. Science Reports 7: 1-10.</p>
<p>Espinosa, C., Garcia Beltran, J.M., Esteban, M.A., and Cuesta, A. 2018. In vitro effects of virgin microplastics on fish head-kidney leucocyte activities. Environmental Pollution 235: 30-38.</p>
<p>Evans, A. R., Limp-Foster, M., & Kelley, M. R. (2000). Going APE over ref-1. Mutation Research/DNA Repair, 461(2), 83-108.</p>
<p>Imhof, H.K., Rusek, J., Thiel, M., Wolinska, J., and Laforsch, C. 2017. Do microplastic particles affect Daphnia magna at the morphological life history and molecular level? Public Library of Science One 12: 1-20.</p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Auten, Richard L., and Jonathan M. Davis. 2009. “Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details.” Pediatric Research 66 (2): 121–27.</span></p>
<p>Jeong, J. and Choi, J. 2020. Development of AOP relevant to microplastics based on toxicity mechanisms of chemical additives using ToxCast™ and deep learning models combined approach. Environment International 137:105557.</p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Tirmenstein, M. A., F. A. Nicholls-Grzemski, J. G. Zhang, and M. W. Fariss. 2000. “Glutathione Depletion and the Production of Reactive Oxygen Species in Isolated Hepatocyte Suspensions.” Chemico-Biological Interactions 127 (3): 201–17.</span></p>
<p>Jeong, C.B., Kang, H.M., Lee, M.C., Kim, D.H., Han, J., Hwang, D.S. Souissi, S., Lee, S.J., Shin, K.H., Park, H.G., and Lee, J.S. 2017. Adverse effects of microplastics and oxidative stress-induced MAPK/NRF2 pathway-mediated defense mechanisms in the marine copepod Paracyclopina nana. Science Reports 7: 1-11.</p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Kalia, Sumiti, and M. P. Bansal. 2008. “Diethyl Maleate-Induced Oxidative Stress Leads to Testicular Germ Cell Apoptosis Involving Bax and Bcl-2.” Journal of Biochemical and Molecular Toxicology 22 (6): 371–81.</span></p>
<p>Jeong, C.B., Wong, E.J., Kang, H.M., Lee, M.C., Hwang, D.S., Hwang, U.K., Zhou, B., Souissi, S., Lee, S.J., and Lee, J.S. 2016. Microplastic size-dependent toxicity, oxidative stress induction, and p-JNK and p-p38 activation in the Monogonout rotifer (Brachionus koreanus). Environmental Science and Technology 50: 8849-8857.</p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Abarikwu, S. O., E. O. Farombi, and A. B. Pant. 2011. “Biflavanone-Kolaviron Protects Human Dopaminergic SH-SY5Y Cells against Atrazine Induced Toxic Insult.” Toxicology in Vitro: An International Journal Published in Association with BIBRA 25 (4): 848–58.</span></p>
<p>Juan, C.A., de la Lastra, J.M.P., Plou, F.J., and Lebena, E.P. 2021. The chemistry of reactive oxygen species (ROS) revisited: Outlining their role in biological macromolecules (DNA, lipids and proteins) and induced pathologies. International Journal of Molecular Sciences 22: 4642.</p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Rizzetti, Danize Aparecida, Caroline Silveira Martinez, Alyne Goulart Escobar, Taiz Martins da Silva, José Antonio Uranga-Ocio, Franck Maciel Peçanha, Dalton Valentim Vassallo, Marta Miguel Castro, and Giulia Alessandra Wiggers. 2017. “Egg White-Derived Peptides Prevent Male Reproductive Dysfunction Induced by Mercury in Rats.” Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 100 (February): 253–64.</span></p>
<p><span style="background-color:#ffffff; color:#222222; font-family:Arial,sans-serif; font-size:13px">Lacy, F., Kailasam, M. T., O’Connor, D. T., Schmid-Schönbein, G. W., & Parmer, R. J. (2000). Plasma hydrogen peroxide production in human essential hypertension: role of heredity, gender, and ethnicity. </span><em>Hypertension</em><span style="background-color:#ffffff; color:#222222; font-family:Arial,sans-serif; font-size:13px">, </span><em>36</em><span style="background-color:#ffffff; color:#222222; font-family:Arial,sans-serif; font-size:13px">(5), 878-884.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">El-Desoky, Gaber E., Samir A. Bashandy, Ibrahim M. Alhazza, Zeid A. Al-Othman, Mourad A. M. Aboul-Soud, and Kareem Yusuf. 2013. “Improvement of Mercuric Chloride-Induced Testis Injuries and Sperm Quality Deteriorations by Spirulina Platensis in Rats.” PloS One 8 (3): e59177.</span></p>
<p>Lei, L., Wu, S., Lu, S., Liu, M., Song, Y., Fu, Z., Shi, H., Raley-Susman, K.M., and He, D. 2018. Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematode Caenorhabditis elegans. Science of the Total Environment 619-620: 1-8.</p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Ma, Yan, Mingkun Zhu, Liping Miao, Xiaoyun Zhang, Xinyang Dong, and Xiaoting Zou. 2018. “Mercuric Chloride Induced Ovarian Oxidative Stress by Suppressing Nrf2-Keap1 Signal Pathway and Its Downstream Genes in Laying Hens.” Biological Trace Element Research 185 (1): 185–96.</span></p>
<p>Marshall, H. E., Merchant, K., & Stamler, J. S. (2000). Nitrosation and oxidation in the regulation of gene expression. The FASEB Journal, 14(13), 1889-1900.</p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Yin, Huiyong, Libin Xu, and Ned A. Porter. 2011. “Free Radical Lipid Peroxidation: Mechanisms and Analysis.” Chemical Reviews 111 (10): 5944–72.</span></p>
<p>Minc, E., De Coppet, P., Masson, P., Thiery, L., Dutertre, S., Amor-Guéret, M., & Jaulin, C. (1999). The human copper-zinc superoxide dismutase gene (SOD1) proximal promoter is regulated by Sp1, Egr-1, and WT1 via non-canonical binding sites. Journal of Biological Chemistry, 274(1), 503-509.</p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Auten, Richard L., and Jonathan M. Davis. 2009. “Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details.” Pediatric Research 66 (2): 121–27.</span></p>
<p>Paul-Pont, I., Lacroix, C., Gonzalez Fernandez, D., Hegaret, H., Lambert, C., Le Goic, N., Frere, L., Cassone, A.L., Sussarellu, R. Fabioux, C., Guyomarch, J., Albentosa, M., Huvet, A., and Soudant, P. 2016. Exposure of marine mussels Mytillus spp. to polystyrene microplastics: Toxicity and influence on fluoranthene bioaccumulation. Environmental Pollution 216: 724-737.</p>
<p>Pines, A., Bivi, N., Romanello, M., Damante, G., Kelley, M. R., Adamson, E. D., ... & Tell, G. (2005). Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: evidence for an autoregulatory loop. Free radical research, 39(3), 269-281.</p>
<p>Ramana, C. V., Boldogh, I., Izumi, T., & Mitra, S. (1998). Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals. Proceedings of the National Academy of Sciences, 95(9), 5061-5066.</p>
<p>Ray, P.D., Huang, B.-W., and Tsuji, Y. 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signalling. Cellular Signalling 24:981-990.</p>
<p>Schrinzi, G.F., Perez-Pomeda, I., Sanchis, J., Rossini, C., Farre, M., and Barcelo, D. 2017. Cytotoxic effects of commonly used nanomaterials and microplastics on cerebral and epithelial human cells. Environmental Research 159: 579-587.</p>
<p>Shields, H.J., Traa, A., and Van Raamsdonk, J.M. 2021. Beneficial and Detrimental Effects of Reactive Oxygen Species on Lifespan: A Comprehensive Review of Comparative and Experimental Studies.</p>
<p>Veneman, W.J., Spaink, H.P., Brun, N.R., Bosker, T., and Vijver, M.G. 2017. Pathway analysis of systemic transcriptome responses to injected polystyrene particles in zebrafish larvae. Aquatic Toxicology 190: 112-120.</p>
<p>Yu, P., Liu, Z., Wu, D., Chen, M., Lv, W., and Zhao, Y. 2018. Accumulation of polystyrene microplastics in juvenile Eriocheir sinensis and oxidative stress effects in the liver. Aquatic Toxicology 200: 28-36.</p>
<p> </p>
<p> </p>
</div>
<div>
<h4><a href="/relationships/3364">Relationship: 3364: Increase, LPO leads to Cell cycle, disrupted</a></h4>
<h4><a href="/relationships/3116">Relationship: 3116: Oxidative Stress leads to Increase, LPO</a></h4>
<td><a href="/aops/332">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/331">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/332">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/331">Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and reduced cell proliferation</a></td>
<td><a href="/aops/333">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
<h4><a href="/relationships/2205">Relationship: 2205: Decrease, Cell proliferation leads to Decrease, Growth</a></h4>
<h4><a href="/relationships/2203">Relationship: 2203: Decrease, Coupling of OXPHOS leads to Decrease, ATP pool</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>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>Moderate</td>
<td>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/290">Mitochondrial ATP synthase antagonism leading to growth inhibition (1)</a></td>
<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/286">Mitochondrial complex III antagonism leading to growth inhibition (1)</a></td>
<td><a href="/aops/324">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/267">Uncoupling of oxidative phosphorylation leading to growth inhibition via glucose depletion</a></td>
<td><a href="/aops/325">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/333">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation</a></td>
<td><a href="/aops/326">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/332">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/331">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/331">Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and reduced cell proliferation</a></td>
<td><a href="/aops/332">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/333">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>adjacent</td>
<td></td>
<td></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>
</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 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>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 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>Relationship 2205 is considered applicable to all lifestages, as cell proliferation leading to growth is essential for maintaining basic biological processes throughout an organism’s life.</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 reduced cell proliferation (cell growth, division or a combination of these) leading to reduced tissue, organ or individual growth.</p>
<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 2205 is considered</strong> moderate.</p>
<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 2205 is considered</strong> high.</p>
<p style="text-align:justify"><strong>The biological plausibility of Relationship 2203 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>
<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 2205 is considered</strong> low.</p>
<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>
<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>
<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">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>
<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>
<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 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>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>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>Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.</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>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>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>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>
<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>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>
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<div>
<h4><a href="/relationships/3603">Relationship: 3603: Decrease, ATP pool leads to Decrease, Cell growth</a></h4>
<td><a href="/aops/325">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/332">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/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>
</tbody>
</table>
</div>
</div>
<div>
<h4><a href="/relationships/3604">Relationship: 3604: Decrease, Cell growth leads to Decrease, Growth</a></h4>
<td><a href="/aops/325">Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/332">Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/600">Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>