<td>Under development: Not open for comment. Do not cite</td>
<td>Under Development: Contributions and Comments Welcome</td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
<h2>Abstract</h2>
<p><span style="font-family:Arial,Helvetica,sans-serif">Here, an Adverse Outcome Pathway (AOP) is proposed for reproductive dysfunction via oxidative stress, which is motivated by the current understanding of the role of oxidative stress in reproductive disorders. The AOP was developed based on OECD's guide no. 184 and the specific considerations of OECD Users' handbook supplement to the guidance document for developing and assessing AOPs (no. 233).</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">According to qualitative and quantitative experimental data that were evaluated, GSH conjugation is the first upstream Key Event (KE) of this AOP, triggering oxidative stress (OS). This event causes depletion of GSH basal levels (KE2). Consequently, this reduction of free GSH induces an increase of ROS (KE3) generated by natural cellular metabolic processes (cellular respiration) of the organisms. As expected, the intensified growth of these reactive species' levels, in turn, induces an increase of lipid peroxidation (KE4). This KE, consequently, leads to a rise in the amount of toxic substances, such as malondialdehyde and hydroxynonenal. Both are intrinsically associated with the decrease in the quality and competence of gamete cell division, and, consequently, cause impairment of fertility (KE5 and Adverse Outcome).</span></p>
</div>
<div id="background">
<h3>Background</h3>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">This AOP was developed for the project "CHRONIC TOXICITY OF PESTICIDES IN DRINKING WATER IN PARAÍBA (TRIGGER): IDENTIFYING THE TRIGGERS OF A SILENT EPIDEMIC," financed by the "Fundação de Apoio à Pesquisa do Estado da Paraíba (FAPESQ-PB)." The project aims to understand how oxidative stress and reproductive toxicity can be triggered in animals by aquatic pollutants, such as atrazine</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Biological plausibility, empirical support and quantitative understanding of the KERs and the evidence that uphold essentialities of KEs in this AOP were analyzed together for the overall assessment of an AOP. In this case, overall assessment (WoE) of the general biological plausibility and of the empirical support of KERs was considered as high for this AOP, as well as essentiality, once for this criterion the first four KEs that trigger the AO are also classified as such. Finally, although the amount of data that support each of the relations differed considerably among them in number, it was possible to obtain an overview about the quantitative comprehension of the KERs, as well as understand their mechanisms. Nevertheless, it is suitable to suggest that more data must be generated, with regard to KER 2879, in order to improve comprehension of this relation among different taxonomic groups.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><strong>Empirical domain of applicability</strong></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><em>Sex<strong>: </strong></em>The AOP is applicable to males and females.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><em>Life stages<strong>: </strong></em>All life stages are relevant to this AOP.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><em>Taxonomic<strong>: </strong></em>The assumed empirical domain of applicability of this AOP is fish and mammals.</span></p>
<p> </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"> <strong>Biologically plausible domain of applicability</strong></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">All the key events described should be conserved among animal species, suggesting that the AOP may also have relevance for amphibians, reptiles, birds and and invertebrates with sexual reproduction. However, interspecies differences are possible because the effectiveness of GSH conjugation as a detoxification mechanism may depend on the species and the specific chemical being considered (Summer et al., 1979).</span></p>
<h3>Essentiality of the Key Events</h3>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">After blocking the synthesis of GSH with the <strong>inhibitor buthionine sulfoximine (BSO)</strong> – at a dose of 2 mmol/kg at 12-hour intervals for 7 days – male rats (4 months old) experienced a dramatic decrease in GSH levels. In the seminal vesicles, there was a depletion of 71% in the content, while in the epididymal tissues, this depletion was more severe: 81% in the caput, 87% in the corpus, and 92% in the cauda of the epididymis. Furthermore, the enzymatic activity of catalase increased significantly in the epididymal tissues, while, on the other hand, the activity of manganese superoxide dismutase (Mn SOD) and glutathione peroxidase (GPX) decreased in the seminal vesicle. Additionally, the <strong>sperm motility</strong> of the animals was <strong>reduced </strong>(Zubkova et al., 2004).</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">In another in vivo study, <strong>the administration of BSO</strong> for 35 days in BALB/c mice at 8 weeks of age – at 2 mmol/kg/day – caused a decrease in GSH content, as well as in catalase (CAT), SOD, and GPX activity. Meanwhile, the MDA content in the testes increased considerably, and a reduction in fertility was recorded through a <strong>decrease in normal sperm and sperm motility and an increase in abnormal sperm</strong> (Sajjadian et al., 2014). Moreover, according to Lopez and Luderer (2004), rats treated with BSO 5 mmol/kg body weight twice a day showed both a decrease in GSH content and an <strong>increase in atretic antral follicles in the ovaries</strong>. On the other hand, rats treated with BSO 4 mmol/kg of body weight twice a day showed significantly decreased levels of GSH and enzymatic activity of CAT, SOD, and GPX in blood and erythrocytes, as well as increased levels of MDA. However, glutathione-monoester therapy during exposure promoted the recovery of levels and activity of these oxidative stress markers in animals treated with BSO (Rajasekaran et al., 2004).</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">In male <strong><em>Nrf2</em><sup>-/-</sup> knockout mice</strong>, there was a reduction in gene expression levels of antioxidant enzymes in the testis and epididymis, including catalytic glutamate cysteine ligase (<em>Gclc</em>), glutamate cysteine ligase modifying subunit (<em>Gclm</em>) – the rate-limiting enzyme in GSH synthesis – glutathione transferase m1 (<em>Gstm1</em>), <em>Gstm2</em>, <em>Gsta3</em>, and <em>Sod2</em>, as well as a depletion in GSH concentration and GPX activity compared to wild-type males. In addition, MDA levels were shown to be significantly increased, while fertility was reduced by the <strong>decrease in the number of litters and pups</strong> (Nakamura et al., 2010). Furthermore, Nakamura et al. (2011) showed that <strong><em>Gclm </em>null female</strong> mice show a decrease in GSH content in ovulated oocytes and a decrease in fertility through the <strong>reduction of litter and offspring production</strong>. Additionally, Lim et al. (2015) found a drop in GSH levels and Nernst potential (Eh) (indicating oxidative stress), an increase in 4-hydroxynonenal (4-HNE), and a <strong>decline in ovarian follicles</strong> in <strong><em>Gclm </em>null female mice</strong>. Besides this, Lim et al. (2020) showed that female mice lacking the <em>Gclm </em>gene show depleted GSH concentrations and a reduction in the number of healthy follicles.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">Moreover, Garratt et al. (2013) showed that <strong><em>Sod1</em><sup>-/-</sup> mice</strong> have <strong>impaired </strong>sperm <strong>motility </strong>and <strong><em>in vivo</em> fertilization</strong> compared to WT animals. Furthermore, Imai et al. (2009) showed that s<strong>permatocyte-specific <em>Gpx4</em><sup>-/-</sup> knockout mice</strong> are completely <strong>infertile</strong>, whereas <em>GPx4</em><sup>+/−</sup> and transgenic rescued <em>Gpx4</em><sup>-/- </sup>knockout mice were fully fertile. Additionally, according to Schneider et al. (2009), <strong><em>mGpx4</em><sup>-/-</sup></strong> (mitochondrial GPx4) <strong>knockout mice</strong> are <strong>infertile </strong>and have less motile and progressive sperm compared to WT.</span></span></p>
<p>Table 2: Summary of <em>in vivo</em> studies with fertility endpoints for chemical inhibitors or gene knockout experiments as evidence to support the essentiality of KEs.</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><span style="font-size:12.0pt">↑ via CAT, GPx and SOD units</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><span style="font-size:12.0pt">↑ via MDA and HAE*</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Several chemicals that undergo GSH conjugation at high concentrations cause depletion of GSH supplies in the liver and other tissues (D’Souza, Francis, and Andersen 1988; D’Souza and Andersen 1988; Csanády et al. 1996; Mulder and Ouwerkerk-Mahadevan 1997; Fennell and Brown 2001).</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Diethyl maleate at 0.1, 0.5, 1, 2.5, and 5 mM for five hours caused GSH depletion in hepatocytes at all concentrations in a dose-dependent manner. However, only 5 mM of the compound was able to consume GSH to the point that this antioxidant was kept below detection levels (4%) and led to overproduction of ROS (Tirmenstein et al. 2000).</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Adult rats treated with BSO 20 and 30 mM for 10 days diligently showed a reduction of, respectively, 44.25% and 60.14% of liver GSH content, while H2O2 levels underwent an augmentation of 42 and 60%, in that order (Ford et al. 2006).</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">For instance, empirical evidence shows 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-family:Arial,Helvetica,sans-serif">Experimental evidence showed that the lipid peroxidation product 4-HNE, at 0, 5, 10, 20, 30, and 50 µM, induces a dose-dependent decrease in meiotic competence during in vitro oocyte maturation, as well as aneuploidies in germinal vesicle (GV) oocytes from 20 µM of 4-HNE (Mihalas et al. 2017).</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">BSO for 35 days in BALB/c mice at 8 weeks of age – at 2 mmol/kg/day – caused a decrease in GSH content, as well as in catalase (CAT), SOD, and GPX activity. Meanwhile, the MDA content in the testes increased considerably, and reduction in fertility was recorded through a decrease in normal sperm and sperm motility and an increase in abnormal sperm (Sajjadian et al., 2014).</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">In male <em>Nrf2</em><sup>-/-</sup> knockout mice, there was a reduction in gene expression levels of antioxidant enzymes in the testis and epididymis, including catalytic glutamate cysteine ligase (<em>Gclc</em>), glutamate cysteine ligase modifying subunit (<em>Gclm</em>) – the rate-limiting enzyme in GSH synthesis – glutathione transferase m1 (<em>Gstm1</em>), <em>Gstm2</em>, <em>Gsta3</em>, and <em>Sod2</em>, as well as a depletion in GSH concentration and GPX activity compared to wild-type males. In addition, MDA levels were shown to be significantly increased, while fertility was reduced by the decrease in the number of litters and pups (Nakamura et al., 2010).</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Lim et al. (2015) found a drop in GSH levels and Nernst potential (Eh) (indicating oxidative stress), an increase in 4-hydroxynonenal (4-HNE), and a decline in ovarian follicles in <em>Gclm </em>null female mice.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">D’Souza, R. W., and M. E. Andersen. 1988. “Physiologically Based Pharmacokinetic Model for Vinylidene Chloride.” Toxicology and Applied Pharmacology 95 (2): 230–40.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">D’Souza, R. W., W. R. Francis, and M. E. Andersen. 1988. “Physiological Model for Tissue Glutathione Depletion and Increased Resynthesis after Ethylene Dichloride Exposure.” The Journal of Pharmacology and Experimental Therapeutics 245 (2): 563–68.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Csanády, G. A., P. E. Kreuzer, C. Baur, and J. G. Filser. 1996. “A Physiological Toxicokinetic Model for 1,3-Butadiene in Rodents and Man: Blood Concentrations of 1,3-Butadiene, Its Metabolically Formed Epoxides, and of Haemoglobin Adducts--Relevance of Glutathione Depletion.” Toxicology 113 (1-3): 300–305.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Mulder, G. J., and S. Ouwerkerk-Mahadevan. 1997. “Modulation of Glutathione Conjugation in Vivo: How to Decrease Glutathione Conjugation in Vivo or in Intact Cellular Systems in Vitro.” Chemico-Biological Interactions 105 (1): 17–34.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Fennell, T. R., and C. D. Brown. 2001. “A Physiologically Based Pharmacokinetic Model for Ethylene Oxide in Mouse, Rat, and Human.” Toxicology and Applied Pharmacology 173 (3): 161–75.</span></p>
<p><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><span style="font-family:Arial,Helvetica,sans-serif">Ford, Rebecca J., Drew A. Graham, Steven G. Denniss, Joe Quadrilatero, and James W. E. Rush. 2006. “Glutathione Depletion in Vivo Enhances Contraction and Attenuates Endothelium-Dependent Relaxation of Isolated Rat Aorta.” Free Radical Biology & Medicine 40 (4): 670–78.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Garratt, M., Bathgate, R., de Graaf, S. P., and Brooks, R. C. 2013. “Copper-zinc superoxide dismutase deficiency impairs sperm motility and in vivo fertility.” Reproduction, 146(4), 297-304.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Schneider, M., Forster, H., Boersma, A., Seiler, A., Wehnes, H., Sinowatz, F., ... and Conrad, M. 2009. “Mitochondrial glutathione peroxidase 4 disruption causes male infertility”. The FASEB journal, 23(9), 3233-3242.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Imai, H., Hakkaku, N., Iwamoto, R., Suzuki, J., Suzuki, T., Tajima, Y., ... and Nakagawa, Y. 2009. “Depletion of selenoprotein GPx4 in spermatocytes causes male infertility in mice”. Journal of Biological Chemistry, 284(47), 32522-32532.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Lim, J., Ali, S., Liao, L. S., Nguyen, E. S., Ortiz, L., Reshel, S., and Luderer, U. 2020. “Antioxidant supplementation partially rescues accelerated ovarian follicle loss, but not oocyte quality, of glutathione-deficient mice.” Biology of Reproduction, 102(5), 1065-1079.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Lim, J., Nakamura, B. N., Mohar, I., Kavanagh, T. J., and Luderer, U. 2015. “Glutamate cysteine ligase modifier subunit (Gclm) null mice have increased ovarian oxidative stress and accelerated age-related ovarian failure.” Endocrinology, 156(9), 3329-3343.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Nakamura, B. N., Lawson, G., Chan, J. Y., Banuelos, J., Cortés, M. M., Hoang, Y. D., ... and Luderer, U. 2010. “Knockout of the transcription factor NRF2 disrupts spermatogenesis in an age-dependent manner. Free Radical Biology and Medicine, 49(9), 1368-1379.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Nakamura, B. N., Fielder, T. J., Hoang, Y. D., Lim, J., McConnachie, L. A., Kavanagh, T. J., and Luderer, U. 2011. Lack of maternal glutamate cysteine ligase modifier subunit (Gclm) decreases oocyte glutathione concentrations and disrupts preimplantation development in mice.” Endocrinology, 152(7), 2806-2815.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Lopez, S. G., and Luderer, U. 2004. “Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis.” Free Radical Biology and Medicine, 36(11), 1366-1377.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Sajjadian, F., Roshangar, L., Hemmati, A., Nori, M., Soleimani-Rad, S., and Soleimani-Rad, J. 2014. “The effect of BSO-induced oxidative stress on histologic feature of testis: testosterone secretion and semen parameters in mice.” Iranian journal of basic medical sciences, 17(8), 606.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Zubkova, E. V., and Robaire, B. 2004. “Effect of glutathione depletion on antioxidant enzymes in the epididymis, seminal vesicles, and liver and on spermatozoa motility in the aging brown Norway rat.” Biology of reproduction, 71(3), 1002-1008.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Rajasekaran, N. S., Devaraj, N. S., and Devaraj, H. 2004. “Modulation of rat erythrocyte antioxidant defense system by buthionine sulfoximine and its reversal by glutathione monoester therapy.’ Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1688(2), 121-129.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Summer, K. H., Rozman, K., Coulston, F., and Greim, H. 1979. Urinary excretion of mercapturic acids in chimpanzees and rats. Toxicology and Applied Pharmacology, 50(2), 207-212.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><strong>Plausible domain of applicability</strong></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><strong><em>Taxonomic applicability</em>: </strong>The GSH conjugation is known to occur in eukaryotic cells.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><strong><em>Life stage applicability</em></strong>: GSH conjugation can be measured at any stage of life.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><em><strong>Sex applicability</strong></em>: GSH conjugation can be measured in both male and female species. </span></span></p>
<h4>Key Event Description</h4>
<p>Glutathione, GSH (γ-L-glutamyl-L-cysteinyl-glycine) is a tripeptide synthesized in the intracellular media in a two-step process: bond between glutamic acid and cysteine by the enzyme glutamate-cystein ligase followed by the combination of the resulting dipeptide with a glycin, which is catalyzed by glutathione-synthetase (Lushchak 2012; Hellou, Ross, and Moon 2012; Aquilano, Baldelli, and Ciriolo 2014). In the oxidative stress pathway, GSH is used as substrate by different types and isoforms of enzymes, such as glutathione-reductases (GRs), glutathione-peroxidases (GPXs) and glutathione-transferases (GSTs).<br />
Conjugation with glutathione might happen spontaneously, but it is a reaction primarily catalyzed by GSTs (X. Li 2009). This class of enzymes conjugates the tripeptide with toxic chemicals (e.g. arene, oxides, unsaturated carbonyls, organic halides) in order to neutralize them, making them harmless to cells through a Michael addition reaction (Forman, Zhang, and Rinna 2009; Lushchak 2012; Aquilano, Baldelli, and Ciriolo 2014). In this case, the sulfhydryl group acts as a nucleophile and binds, for instance, to an amine group or to an atom such as Cl, as well as attacks electrophilic sites of xenobiotics (X. Li 2009). Conjugates generated from this reaction, overall, are less toxic or are excreted from cells, which causes GSH depletion (Forman, Zhang, and Rinna 2009).</p>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify">Liquid chromatography–mass spectrometry (Pallante et al. 1986; Plakunov et al. 1987; Pflugmacher et al. 1998; Wiegand et al. 2001a; Dai et al. 2008; Dionisio, Gautam, and Fomsgaard 2019).</p>
<h4>References</h4>
<p>Lushchak, Volodymyr I. 2012. “Glutathione Homeostasis and Functions: Potential Targets for Medical Interventions.” Journal of Amino Acids 2012 (February): 736837.</p>
<p>Hellou, Jocelyne, Neil W. Ross, and Thomas W. Moon. 2012. “Glutathione, Glutathione S-Transferase, and Glutathione Conjugates, Complementary Markers of Oxidative Stress in Aquatic Biota.” Environmental Science and Pollution Research International 19 (6): 2007–23.</p>
<p>Aquilano, Katia, Sara Baldelli, and Maria R. Ciriolo. 2014. “Glutathione: New Roles in Redox Signaling for an Old Antioxidant.” Frontiers in Pharmacology 5 (August): 196.</p>
<p>Forman, Henry Jay, Hongqiao Zhang, and Alessandra Rinna. 2009. “Glutathione: Overview of Its Protective Roles, Measurement, and Biosynthesis.” Molecular Aspects of Medicine 30 (1-2): 1–12.</p>
<p>Li, Xianchun. 2009. “Glutathione and Glutathione-S-Transferase in Detoxification Mechanisms.” In General, Applied and Systems Toxicology. Chichester, UK: John Wiley & Sons, Ltd. https://doi.org/10.1002/9780470744307.gat166.</p>
<p>Pallante, S. L., C. A. Lisek, D. M. Dulik, and C. Fenselau. 1986. “Glutathione Conjugates. Immobilized Enzyme Synthesis and Characterization by Fast Atom Bombardment Mass Spectrometry.” Drug Metabolism and Disposition: The Biological Fate of Chemicals 14 (3): 313–18.</p>
<p>Plakunov, I., T. A. Smolarek, D. L. Fischer, J. C. Wiley Jr, and W. M. Baird. 1987. “Separation by Ion-Pair High-Performance Liquid Chromatography of the Glucuronide, Sulfate and Glutathione Conjugates Formed from Benzo[a]pyrene in Cell Cultures from Rodents, Fish and Humans.” Carcinogenesis 8 (1): 59–66.</p>
<p>Pflugmacher, S., C. Wiegand, A. Oberemm, K. A. Beattie, E. Krause, G. A. Codd, and C. E. Steinberg. 1998. “Identification of an Enzymatically Formed Glutathione Conjugate of the Cyanobacterial Hepatotoxin Microcystin-LR: The First Step of Detoxication.” Biochimica et Biophysica Acta 1425 (3): 527–33.</p>
<p>Wiegand, C., E. Krause, C. Steinberg, and S. Pflugmacher. 2001a. “Toxicokinetics of Atrazine in Embryos of the Zebrafish (Danio Rerio).” Ecotoxicology and Environmental Safety 49 (3): 199–205.</p>
<p>Dai, Ming, Ping Xie, Gaodao Liang, Jun Chen, and Hehua Lei. 2008. “Simultaneous Determination of Microcystin-LR and Its Glutathione Conjugate in Fish Tissues by Liquid Chromatography-Tandem Mass Spectrometry.” Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences 862 (1-2): 43–50.</p>
<p>Dionisio, Giuseppe, Maheswor Gautam, and Inge Sindbjerg Fomsgaard. 2019. “Identification of Azoxystrobin Glutathione Conjugate Metabolites in Maize Roots by LC-MS.” Molecules 24 (13). https://doi.org/10.3390/molecules24132473.</p>
<p><strong>Plausible domain of applicability</strong></p>
<p><strong><em>Taxonomic applicability</em>: </strong>The GSH depletion is known to occur in eukaryotic cells.</p>
<p><strong><em>Life stage applicability</em></strong>: GSH depletion can be measured at any stage of life.</p>
<p><em><strong>Sex applicability</strong></em>: GSH depletion can be measured in both male and female species. </p>
<h4>Key Event Description</h4>
<p>GSH depletion is commonly observed in different types of organs and cells (Deneke and Fanburg 1989; Lushchak 2012; Aquilano, Baldelli, and Ciriolo 2014). One of the main roles of this antioxidant is to sequester free radicals in order to prevent cell damage. A decline in GSH levels has been thoroughly related to the increase of reactive oxygen species, as well as to lipid peroxides, culminating in tissue oxidative stress (Comporti et al. 1991; Martin and Teismann 2009; Lushchak 2012; Aquilano, Baldelli, and Ciriolo 2014). </p>
<h4>How it is Measured or Detected</h4>
<ul>
<li>Photocolorimetric assays (Rahman 2007; Massarsky, Kozal, and Di Giulio 2017),</li>
<li>HPLC (Afzal et al. 2002; J. Liu et al. 2010) </li>
<li>Through commercial kits purchased from specialized companies.</li>
</ul>
<h4>References</h4>
<p>Deneke, S. M., and B. L. Fanburg. 1989. “Regulation of Cellular Glutathione.” The American Journal of Physiology 257 (4 Pt 1): L163–73.</p>
<p>Lushchak, Volodymyr I. 2012. “Glutathione Homeostasis and Functions: Potential Targets for Medical Interventions.” Journal of Amino Acids 2012 (February): 736837.</p>
<p>Aquilano, Katia, Sara Baldelli, and Maria R. Ciriolo. 2014. “Glutathione: New Roles in Redox Signaling for an Old Antioxidant.” Frontiers in Pharmacology 5 (August): 196.</p>
<p>Comporti, M., E. Maellaro, B. Del Bello, and A. F. Casini. 1991. “Glutathione Depletion: Its Effects on Other Antioxidant Systems and Hepatocellular Damage.” Xenobiotica; the Fate of Foreign Compounds in Biological Systems 21 (8): 1067–76.</p>
<p>Martin, Heather L., and Peter Teismann. 2009. “Glutathione--a Review on Its Role and Significance in Parkinson’s Disease.” FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 23 (10): 3263–72.</p>
<p>Rahman, Khalid. 2007. “Studies on Free Radicals, Antioxidants, and Co-Factors.” Clinical Interventions in Aging 2 (2): 219–36.</p>
<p>Massarsky, Andrey, Jordan S. Kozal, and Richard T. Di Giulio. 2017. “Glutathione and Zebrafish: Old Assays to Address a Current Issue.” Chemosphere 168 (February): 707–15.</p>
<p>Afzal, Mohammed, Aqeela Afzal, Andrew Jones, and Donald Armstrong. 2002. “A Rapid Method for the Quantification of GSH and GSSG in Biological Samples.” Methods in Molecular Biology 186: 117–22.</p>
<p>Liu, Jiaofang, Chunyan Bao, Xinhua Zhong, Chunchang Zhao, and Linyong Zhu. 2010. “Highly Selective Detection of Glutathione Using a Quantum-Dot-Based OFF–ON Fluorescent Probe.” Chemical Communications 46 (17): 2971–73</p>
<td><a href="/aops/383">Aop:383 - Inhibition of Angiotensin-converting enzyme 2 leading to liver fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/382">Aop:382 - Angiotensin II type 1 receptor (AT1R) agonism leading to lung fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/384">Aop:384 - Hyperactivation of ACE/Ang-II/AT1R axis leading to chronic kidney disease </a></td>
<td>KeyEvent</td>
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<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
</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/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/327">Aop:327 - Excessive reactive oxygen species production leading to mortality (1)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/328">Aop:328 - Excessive reactive oxygen species production leading to mortality (2)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/329">Aop:329 - Excessive reactive oxygen species production leading to mortality (3)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/330">Aop:330 - Excessive reactive oxygen species production leading to mortality (4)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/26">Aop:26 - Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/534">Aop:534 - Succinate dehydrogenase (SDH) inhibition leads to cancer through oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/273">Aop:273 - Mitochondrial complex inhibition leading to liver injury</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/488">Aop:488 - Increased reactive oxygen species production leading to decreased cognitive function</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/298">Aop:298 - Increase in reactive oxygen species (ROS) leading to human treatment-resistant gastric cancer</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/27">Aop:27 - Cholestatic Liver Injury induced by Inhibition of the Bile Salt Export Pump (ABCB11)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/511">Aop:511 - The AOP framework on ROS-mediated oxidative stress induced vascular disrupting effects </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/207">Aop:207 - NADPH oxidase and P38 MAPK activation leading to reproductive failure in Caenorhabditis elegans</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/423">Aop:423 - Toxicological mechanisms of hepatocyte apoptosis through the PARP1 dependent cell death pathway </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/481">Aop:481 - AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/282">Aop:282 - Adverse outcome pathway on photochemical toxicity initiated by light exposure</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/569">Aop:569 - Decreased DNA methylation of FAM50B/PTCHD3 leading to IQ loss of children via PI3K-Akt pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/613">Aop:613 - Peroxisome proliferator-activated receptor alpha activation leading to early life stage mortality via increased reactive oxygen species production</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/622">Aop:622 - Calcineurin inhibitor induced nephrotoxicity leading to kidney failure</a></td>
<p>ROS is a normal constituent found in all organisms.</p>
<p>ROS is a normal constituent found in all organisms, <em>lifestages, and sexes.</em></p>
<h4>Key Event Description</h4>
<p>Biological State: increased reactive oxygen species (ROS)</p>
<p>Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.</p>
<p>Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
<p>Reactive oxygen species (ROS) are O<sub>2</sub>- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). </p>
<div>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Reactive oxygen species (ROS) refers to the chemical species superoxide, hydrogen peroxide, and their secondary reactive products. In the biological context, ROS are signaling molecules with important roles in cell energy metabolism, cell proliferation, and fate. Therefore, balancing ROS levels at the cellular and tissue level is an important part of many biological processes. Disbalance, mainly an increase in ROS levels, can cause cell dysfunction and irreversible cell damage.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are produced from both exogenous stressors and normal endogenous cellular processes, such as the mitochondrial electron transport chain (ETC). Inhibition of the ETC can result in the accumulation of ROS. Exposure to chemicals, heavy metal ions, or ionizing radiation can also result in increased production of ROS. Chemicals and heavy metal ions can deplete cellular antioxidants reducing the cell’s ability to control cellular ROS and resulting in the accumulation of ROS. Cellular antioxidants include glutathione (GSH), protein sulfhydryl groups, superoxide dismutase (SOD). </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ROS are radicals, ions, or molecules that have a single unpaired electron in their outermost shell of electrons, which can be categorized into two groups: free oxygen radicals and non-radical ROS [Liou et al., 2010]. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">highly reactive lipid- or carbohydrate-derived carbonyl compounds</span></span></p>
</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>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>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>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>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>Bisht, Shilpa, Muneeb Faiq, Madhuri Tolahunase, and Rima Dada. 2017. “Oxidative Stress and Male Infertility.” Nature Reviews. Urology 14 (8): 470–85.</p>
<p>PCPC. PCPC 2014 safety evaluation guidelines; Chapter 7: Evaluation of Photoirritation and Photoallergy potential. Personal Care Products Council; 2014.</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>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>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>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/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/615">Aop:615 - Suppression of Keap1 cysteine oxidation leading to liver inflammation</a></td>
<p dir="ltr">ROS is a normal constituent found in all organisms, therefore, all organisms containing lipid membranes may be affected by lipid peroxidation. </p>
<p>Structure: Regardless of sex or life stage, when exposed to free radicals, there is potential for lipid peroxidation as a auxiliary response where there are lipid membranes.</p>
<h4>Key Event Description</h4>
<p>Lipid peroxidation is the direct damage to lipids in the membrane of the cell or the membranes of the organelles inside the cells. Ultimately the membranes will break due to the build-up damage in the lipids. This is mainly caused by oxidants which attack lipids specifically, since these contain carbon-carbon double bonds. During lipid peroxidation several lipid radicals are formed in a chain reaction. These reactions can interfere and stimulate each other. Antioxidants, such as vitamin E, can react with lipid peroxy radicals to prevent further damage in the cell (Cooley et al. 2000).</p>
<h4>How it is Measured or Detected</h4>
<p>The main product of lipid peroxidation, malondialdehyde and 4-hydroxyalkenals, is used to measure the degree of this process. This is measured by photocolorimetric assays, quantification of fatty acids by gaseous liquid chromatography (GLC) or high performance (HPLC) (L. Li et al. 2019; Jin et al. 2010a) or through commercial kits purchased from specialized companies.</p>
<p> </p>
<h4>References</h4>
<p>Cooley HM, Evans RE, Klaverkamp JF. 2000. Toxicology of dietary uranium in lake whitefish (Coregonus clupeaformis). Aquatic Toxicology. 48(4):495–515. https://doi.org/10.1016/S0166-445X(99)00057-0</p>
<p>Jin, Yuanxiang, Xiangxiang Zhang, Linjun Shu, Lifang Chen, Liwei Sun, Haifeng Qian, Weiping Liu, and Zhengwei Fu. 2010a. “Oxidative Stress Response and Gene Expression with Atrazine Exposure in Adult Female Zebrafish (Danio Rerio).” Chemosphere 78 (7): 846–52.</p>
<p>Li, Luxiao, Shanshan Zhong, Xia Shen, Qiujing Li, Wenxin Xu, Yongzhen Tao, and Huiyong Yin. 2019. “Recent Development on Liquid Chromatography-Mass Spectrometry Analysis of Oxidized Lipids.” Free Radical Biology & Medicine 144 (November): 16–34.</p>
<td><a href="/aops/7">Aop:7 - Aromatase (Cyp19a1) reduction leading to impaired fertility in adult female</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/51">Aop:51 - PPARα activation leading to impaired fertility in adult male rodents </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/18">Aop:18 - PPARα activation in utero leading to impaired fertility in males</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/64">Aop:64 - Glucocorticoid Receptor (GR) Mediated Adult Leydig Cell Dysfunction Leading to Decreased Male Fertility</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/348">Aop:348 - Inhibition of 11β-Hydroxysteroid Dehydrogenase leading to decreased population trajectory </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/349">Aop:349 - Inhibition of 11β-hydroxylase leading to decresed population trajectory </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/398">Aop:398 - Inhibition of ALDH1A (RALDH) leading to impaired fertility via disrupted meiotic initiation of fetal oogonia of the ovary</a></td>
<td><a href="/aops/398">Aop:398 - Decreased ALDH1A (RALDH) activity leading to decreased fertility via disrupted meiotic initiation of fetal oogonia </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/492">Aop:492 - Glutathione conjugation leading to reproductive dysfunction via oxidative stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/345">Aop:345 - Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/592">Aop:592 - DBDPE-induced DNA strand breaks and LDH activity inhibition leading to population growth rate decline via energy metabolism disrupt and apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/400">Aop:400 - Inhibition of CYP26B1 activity in fetal testis leading to reduced fertility</a></td>
<p><strong>Plausible domain of applicability</strong></p>
<p><strong><em>Taxonomic applicability</em>: </strong>The impaired fertility may also have relevance for fish, mammals, amphibians, reptiles, birds and and invertebrates with sexual reproduction.</p>
<p><strong><em>Life stage applicability</em></strong>: The impaired fertility can be measured at juveniles and adults.</p>
<p><em><strong>Sex applicability</strong></em>: The impaired fertility can be measured in both male and female species. </p>
<h4>Key Event Description</h4>
<p><strong>Biological state</strong></p>
<p>capability to produce offspring</p>
<p><strong>Biological compartments</strong></p>
<p>System</p>
<p><strong>General role in biology</strong></p>
<p>Fertility is the capacity to conceive or induce conception. Impairment of fertility represents disorders of male or female reproductive functions or capacity.</p>
<h4>How it is Measured or Detected</h4>
<p>As a measure, fertility rate, is the number of offspring born per mating pair, individual or population.</p>
<h4>Regulatory Significance of the AO</h4>
<p>Under REACH, information on reproductive toxicity is required for chemicals with an annual production/importation volume of 10 metric tonnes or more. Standard information requirements include a screening study on reproduction toxicity (OECD TG 421/422) at Annex VIII (10-100 t.p.a), a prenatal developmental toxicity study (OECD 414) on a first species at Annex IX (100-1000 t.p.a), and from March 2015 the OECD 443(Extended One-Generation Reproductive Toxicity Study) is reproductive toxicity requirement instead of the two generation reproductive toxicity study (OECD TG 416). If not conducted already at Annex IX, a prenatal developmental toxicity study on a second species at Annex X (≥ 1000 t.p.a.).</p>
<p>Under the Biocidal Products Regulation (BPR), information is also required on reproductive toxicity for active substances as part of core data set and additional data set (EU 2012, ECHA 2013). As a core data set, prenatal developmental toxicity study (EU TM B.31) in rabbits as a first species and a two-generation reproduction toxicity study (EU TM B.31) are required. OECD TG 443 (Extended One-Generation Reproductive Toxicity Study) shall be considered as an alternative approach to the multi-generation study.) According to the Classification, Labelling and Packaging (CLP) regulation (EC, 200; Annex I: 3.7.1.1): a) “reproductive toxicity” includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring; b) “effects on fertility” includes adverse effects on sexual function and fertility; and c) “developmental toxicity” includes adverse effects on development of the offspring.</p>
<h4>References</h4>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2001), <em>Test No. 416: Two-Generation Reproduction Toxicity</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264070868-en">https://doi.org/10.1787/9789264070868-en</a>.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2018), <em>Test No. 443: Extended One-Generation Reproductive Toxicity Study</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264185371-en">https://doi.org/10.1787/9789264185371-en</a>.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2018), <em>Test No. 414: Prenatal Developmental Toxicity Study</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264070820-en">https://doi.org/10.1787/9789264070820-en</a>.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">OECD (2018), "Reproduction/Developmental Toxicity Screening Test (OECD TG 421) and Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422)", in <em>Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption</em>, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264304741-25-en">https://doi.org/10.1787/9789264304741-25-en</a>.</span></span></p>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2877">Relationship: 2877: Conjugation, Glutathione leads to Depletion, GSH</a></h4>
<h4><a href="/relationships/2877">Relationship: 2877: Conjugation, GSH leads to Depletion, GSH</a></h4>
<p>Considering the empirical domain of the evidence, the GSH conjugation leading to GSH depletion is known to occur in all vertebrates animals, but, based on scientific reasoning, it can also occur in eukaryotic organisms in general. It can be measured at any stage of life and in both male and female species.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">GSH is an antioxidant generated in various kinds of cells, however, in vertebrates, this takes place mainly in liver cells, from where it is exported to other cell types (Lu 2013). Around 85% of free GSH is found in the cytoplasm, from where it is distributed to organelles such as mitochondria, which stores approximately 10% of the total GSH content, endoplasmic reticulum and extracellular space (Lu 2013; Aquilano, Baldelli, and Ciriolo 2014). Depletion of free GSH content happens because of the sulfhydryl group of the cysteine residue of this tripeptide reacts with xenobiotics during detoxification process, producing conjugates, which are secreted directly into the bile or converted to mercapturic acids and excreted into the urine, as well as due to the reaction with other reactive species as ROS. Nevertheless, unlike what happens to glycine and glutamate residues with oxidized glutathione (GSSG), which are recycled respectively from detoxification of xenobiotics and ROS-mediated oxidation, the cysteine molecule from GSH is excreted from the organism as a byproduct conjugated to the toxic molecule, causing, thereby, reduction of cellular levels of this limiting amino acid for the tripeptide production. In this way, restoration of regular intracellular GSH levels, via de novo synthesis and still from the reaction of reduction of oxidized glutathione (GSSG) ends up being hampered (X. Li 2009; Lushchak 2012; Gupta 2016; Aquilano, Baldelli, and Ciriolo 2014) and GSH levels are, consequently, depleted.</span></p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">In vertebrate animals, chemicals such as ATZ (Egaas et al. 1993; Wiegand et al. 2001; Elia, Waller, and Norton 2002; Abel et al. 2004; McMullin et al. 2007; LeBlanc and Sleno 2011), DEM (Combes and Backof 1982; Kubal et al. 1995) and Hg (Stricks and Kolthoff 1953; Valko, Morris, and Cronin 2005) are metabolized by GST through reduced GSH-binding in phase II of biotransformation, generating GSH conjugates.</span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif"><em>In vitro</em> and <em>in vivo</em> data reveal that ATZ leads to GSH depletion through that pathway in various fish species. For instance, 25 μg/mL of this chemical uses up GSH neutrophil in common carp after 2 – 3 h of exposure (Wang et al. 2019). In young Prochilodus lineatus, after 24h of exposure at the concentrations of 2 and 10 μg/L, this herbicide did not display any effect on hepatic GSH levels, but, after 48 h, ATZ induced a significant decrease in the content of this biomarker (Santos and Martinez 2012). And in studies of acute toxicity, this chemical caused depletion of GSH level in both young catfish (Rhamdia quelen) (Mela et al. 2013) and in embryos of zebrafish (Danio rerio) (Adeyemi, da Cunha Martins-Junior, and Barbosa 2015) after 96 h of exposure, at the concentrations of 100 μg/L and 0.1 mM, respectively. Confirming these data, at longer exposures in adult female zebrafish, ATZ also underwent a decrease in ovary and liver GSH levels at concentrations above 1 and 10 μg/L, respectively, after 14 days of exposure (Jin et al. 2010). Similarly, common carp submitted to 1/5 of LC50 (96-h) of ATZ for 40 days, showed GSH levels significantly (p < 0.05) reduced in liver cells (Toughan et al. 2018). </span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">As in fishes, this drop in GSH levels is also observed in different organs and tissues of mammals. Albino male rats orally treated with ATZ (200 mg/Kg of body weight/day), for a period of 30 days, exhibited a decrease in brain, hippocampus and submandibular salivary gland GSH contents (Ahmed et al. 2022). Moreover, Sprague-Dawley male rats ATZ-exposed via gavage, for 30 days, showed reduction of total antioxidant capacity in a dose-dependent manner, as well as a significant decrease of free GSH level in testicles of these animals (Song et al. 2014). </span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">GSH-depleting agent DEM, likewise, is able to induce a drop in testicular GSH in BALB/c mice. 52 μM of this chemical intraperitoneally injected, during two weeks, leads to a significant reduction of free GSH levels in testicles of these animals (Kalia and Bansal 2008). (Kaur, Kalia, and Bansal 2006) had previously found evidences of relevant diminishment (p < 0.001) of GSH content and elevation of GSSG levels in testicles of this same animal strain daily submitted to DEM intraperitoneal injection, at 8.7 μM, for two weeks. </span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">Regarding Hg, several taxons also have their GSH levels affected in organs and varied tissues. Adult female zebrafish exposed to 15 and 30 μg/L for a period of 30 days, exhibited a reduction of GSH content in ovaries in a dose-dependent manner (Zhang et al. 2016). In male albino Wistar rats, a single dose (5 mg/Kg bw) of a mercury (II) chloride (HgCl2) solution subcutaneously administered, three times a week, for 60 days, was also able to negatively change GSH testicular content (El-Desoky et al. 2013). Nevertheless, the authors also noticed that animals treated with Spirulina platensis (300 mg/Kg bw), by gavage for 10 consecutive days, before mercury (II) chloride administration and continued up to 60 days along with HgCl2, did not suffer changes in GSH levels, emphasizing downstream KE essentiality, once this can be prevented. This GSH reduction is also seen in bird for Hg. Hy-Line Brown laying hens fed with four experimental diets containing gradual levels of mercury at 0.280, 3.325, 9.415 e 27.240 mg/Kg, respectively, for a period of 10 weeks, displayed GSH content considerably decreased in all Hg-treated groups (Ma et al. 2018).</span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">Hence, it is noted that GSH depletion caused by chemicals happens in all stages of live in teleosts, as well as adult mammals and birds, showing that this KER is conserved among these taxa, which is expected, since this antioxidant participates in basic cellular processes in vertebrate organisms. However, the time necessary for this response varies depending on the different stages and among species, as well as it is dependent on the dose/concentration applied. Still, this is not surprising, because toxicokinetics for chemicals obviously differs among taxa and depends on some variables, such as uptake and solubility.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">GSH depletion depends on the constant conjugation rate of GSH to a xenobiotic, from the initial GSH concentration and its synthesis and degradation rates. Several chemicals that undergo GSH conjugation at high concentrations cause depletion of GSH supplies in the liver and others tissues (D’Souza, Francis, and Andersen 1988; D’Souza and Andersen 1988; Csanády et al. 1996; Mulder and Ouwerkerk-Mahadevan 1997; Fennell and Brown 2001).</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">In this context, the global kinetic equation for GSH consumption through conjugation to xenobiotics, catalyzed by microsomal glutathione transferase 1 (mGST1), purified from rat liver can be defined by (Spahiu et al. 2017) (figure below.). In this equation, C is the electrophilic substrate, while E represents the enzyme and P serves as a GSH-conjugate. In relation to constants, k<sub>2</sub> is the rate for thiolate anion, k<sub>-2</sub> is the rate for the reverse process of thiolate anion, k<sub>3</sub> is the rate for the chemical step that is essentially irreversible, K<sub>C </sub>is the dissociation constant for electrophilic substrate and K<sub>G</sub> is the dissociation constant for GSH (Spahiu et al. 2017). </span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Moreover, thiolate anion formation (k<sub>obs</sub>) can be easily calculated through equations described by (Morgenstern et al. 2001). Kinetic parameters K<sub>M</sub> e k<sub>cat</sub> values for both electrophiles and GSH can also be determined according to the equations established by (Spahiu et al. 2017).Furthermore, nucleophilic reactivity (N) and electrophilicity (E) parameters of GSH have also been settled to a variety of Michael acceptors (Mayer and Ofial 2019). </span></p>
<strong>Response-response relationship</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Velocity of conjugation, however, depends on the kind of GST involved and on the chemical, as well as the organism in which it takes place. For instance, the ATZ-GSH conjugate formed in GSTs from zebrafish embryos works in a time-dependent manner, although conjugation in the microsomal GST increased linearly by a factor of 23 up to 12 h of incubation time, whereas in the soluble GST the conversion rate increased more slowly and was higher by a factor of 5.8 after 24 h of incubation time than that at start (Wiegand et al. 2001). In rats, the estimated GSH conjugation rate constant with ATZ was 0.53 L/mmol/h, a value comparable to that for other chemicals that are largely conjugated by GSTs, even so less than known depleters such as ethylene dichloride (1.2 L/mmol/h) and allyl chloride (9.0 L/mmol/h). Although ATZ is mostly metabolized by GSH, the model estimated that 50% depletion of GSH is predicted to occur, but only after three daily doses of 500 mg ATZ/Kg (McMullin et al. 2003).</span></p>
<strong>Time-scale</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">In humans, intrahepatic glutathione concentration is predicted to be the lowest one, due to conjugation to the reactive intermediate NAPQI, at 6 h after 2 g of intravenous (IV) infusion administration of paracetamol and then to recover slowly. In addition, it responds in a time-dependent way. However, concentrations of glutathione were predicted to be markedly and progressively depleted when patients had an initial 2 g dose and then 1 g dose every 6 h (Geenen et al. 2013).</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">(Hughes, Miller, and Swamidass 2015), for example, constructed a model to predict the GSH reactivity to 1213 molecules and determined the percent depletion of GSH after 15 min incubation with each molecule. In this context, such a model can be easily used for investigation and initial selection of molecules that might impair fertility.</span></p>
<td><span style="font-family:Arial,Helvetica,sans-serif">Singh, Sandhir, and Kiran 2010</span></td>
</tr>
</tbody>
</table>
</div>
<h4>References</h4>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Lu, Shelly C. 2013. “Glutathione Synthesis.” Biochimica et Biophysica Acta 1830 (5): 3143–53.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Aquilano, Katia, Sara Baldelli, and Maria R. Ciriolo. 2014. “Glutathione: New Roles in Redox Signaling for an Old Antioxidant.” Frontiers in Pharmacology 5 (August): 196.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Li, Xianchun. 2009. “Glutathione and Glutathione-S-Transferase in Detoxification Mechanisms.” In General, Applied and Systems Toxicology. Chichester, UK: John Wiley & Sons, Ltd. https://doi.org/10.1002/9780470744307.gat166.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Lushchak, Volodymyr I. 2012. “Glutathione Homeostasis and Functions: Potential Targets for Medical Interventions.” Journal of Amino Acids 2012 (February): 736837.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Gupta, P. K. 2016. “Chapter 8 - Biotransformation.” In Fundamentals of Toxicology, edited by P. K. Gupta, 73–85. Academic Press.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Egaas, E., J. U. Skaare, N. O. Svendsen, M. Sandvik, J. G. Falls, W. C. Dauterman, T. K. Collier, and J. Netland. 1993. “A Comparative Study of Effects of Atrazine on Xenobiotic Metabolizing Enzymes in Fish and Insect, and of the Invitro Phase II Atrazine Metabolism in Some Fish, Insects, Mammals and One Plant Species.” Comparative Biochemistry and Physiology. Part C, Pharmacology, Toxicology & Endocrinology 106 (1): 141–49.</span></span></p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Elia, A. C., W. T. Waller, and S. J. Norton. 2002. “Biochemical Responses of Bluegill Sunfish (Lepomis Macrochirus, Rafinesque) to Atrazine Induced Oxidative Stress.” Bulletin of Environmental Contamination and Toxicology 68 (6): 809–16.</span></span></p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">McMullin, Tami S., William H. Hanneman, Brian K. Cranmer, John D. Tessari, and Melvin E. Andersen. 2007. “Oral Absorption and Oxidative Metabolism of Atrazine in Rats Evaluated by Physiological Modeling Approaches.” Toxicology 240 (1-2): 1–14.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">LeBlanc, André, and Lekha Sleno. 2011. “Atrazine Metabolite Screening in Human Microsomes: Detection of Novel Reactive Metabolites and Glutathione Adducts by LC-MS.” Chemical Research in Toxicology 24 (3): 329–39.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Combes, B., and B. Backof. 1982. “Effect of Diethyl Maleate on the Biliary Excretion Rate of Infused Sulfobromophthalein-Glutathione.” Biochemical Pharmacology 31 (16): 2669–74.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Kubal, G., D. J. Meyer, R. E. Norman, and P. J. Sadler. 1995. “Investigations of Glutathione Conjugation in Vitro by 1H NMR Spectroscopy. Uncatalyzed and Glutathione Transferase-Catalyzed Reactions.” Chemical Research in Toxicology 8 (5): 780–91.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Stricks, W., and I. M. Kolthoff. 1953. “Reactions between Mercuric Mercury and Cysteine and Glutathione. Apparent Dissociation Constants, Heats and Entropies of Formation of Various Forms of Mercuric Mercapto-Cysteine and -Glutathione.” Journal of the American Chemical Society 75 (22): 5673–81.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><a href="http://paperpile.com/b/LRH3Mr/eknN4"><span style="color:#000000">Valko, M., H. Morris, and M. T. D. Cronin. 2005. “Metals, Toxicity and Oxidative Stress.” </span></a><a href="http://paperpile.com/b/LRH3Mr/eknN4"><span style="color:#000000"><em>Current Medicinal Chemistry</em></span></a><a href="http://paperpile.com/b/LRH3Mr/eknN4"><span style="color:#000000"> 12 (10): 1161–1208.</span></a></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Wang, Shengchen, Qiaojian Zhang, Shufang Zheng, Menghao Chen, Fuqing Zhao, and Shiwen Xu. 2019. “Atrazine Exposure Triggers Common Carp Neutrophil Apoptosis via the CYP450s/ROS Pathway.” Fish & Shellfish Immunology 84 (January): 551–57.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Santos, Thais G., and Cláudia B. R. Martinez. 2012. “Atrazine Promotes Biochemical Changes and DNA Damage in a Neotropical Fish Species.” Chemosphere 89 (9): 1118–25.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Mela, M., I. C. Guiloski, H. B. Doria, M. A. F. Randi, C. A. de Oliveira Ribeiro, L. Pereira, A. C. Maraschi, V. Prodocimo, C. A. Freire, and H. C. Silva de Assis. 2013. “Effects of the Herbicide Atrazine in Neotropical Catfish (Rhamdia Quelen).” Ecotoxicology and Environmental Safety 93 (July): 13–21.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Adeyemi, Joseph A., Airton da Cunha Martins-Junior, and Fernando Barbosa Jr. 2015. “Teratogenicity, Genotoxicity and Oxidative Stress in Zebrafish Embryos (Danio Rerio) Co-Exposed to Arsenic and Atrazine.” Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP 172-173 (April): 7–12.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">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.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Toughan, Hosam, Samah R. Khalil, Ashraf Ahmed El-Ghoneimy, Ashraf Awad, and A. Sh Seddek. 2018. “Effect of Dietary Supplementation with Spirulina Platensis on Atrazine-Induced Oxidative Stress- Mediated Hepatic Damage and Inflammation in the Common Carp (Cyprinus Carpio L.).” Ecotoxicology and Environmental Safety 149 (March): 135–42.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Ahmed, Yasmine H., Huda O. AbuBakr, Ismail M. Ahmad, and Zainab Sabry Othman Ahmed. 2022. “Histopathological, Immunohistochemical, And Molecular Alterations In Brain Tissue And Submandibular Salivary Gland Of Atrazine-Induced Toxicity In Male Rats.” Environmental Science and Pollution Research International 29 (20): 30697–711.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Song, Yang, Zhen Chao Jia, Jin Yao Chen, Jun Xiang Hu, and Li Shi Zhang. 2014. “Toxic Effects of Atrazine on Reproductive System of Male Rats.” Biomedical and Environmental Sciences: BES 27 (4): 281–88.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">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></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Kaur, Parminder, Sumiti Kalia, and Mohinder P. Bansal. 2006. “Effect of Diethyl Maleate Induced Oxidative Stress on Male Reproductive Activity in Mice: Redox Active Enzymes and Transcription Factors Expression.” Molecular and Cellular Biochemistry 291 (1-2): 55–61.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Zhang, Qun-Fang, Ying-Wen Li, Zhi-Hao Liu, and Qi-Liang Chen. 2016. “Reproductive Toxicity of Inorganic Mercury Exposure in Adult Zebrafish: Histological Damage, Oxidative Stress, and Alterations of Sex Hormone and Gene Expression in the Hypothalamic-Pituitary-Gonadal Axis.” Aquatic Toxicology 177 (August): 417–24.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">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></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">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></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">D’Souza, R. W., and M. E. Andersen. 1988. “Physiologically Based Pharmacokinetic Model for Vinylidene Chloride.” Toxicology and Applied Pharmacology 95 (2): 230–40.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">D’Souza, R. W., W. R. Francis, and M. E. Andersen. 1988. “Physiological Model for Tissue Glutathione Depletion and Increased Resynthesis after Ethylene Dichloride Exposure.” The Journal of Pharmacology and Experimental Therapeutics 245 (2): 563–68.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Mulder, G. J., and S. Ouwerkerk-Mahadevan. 1997. “Modulation of Glutathione Conjugation in Vivo: How to Decrease Glutathione Conjugation in Vivo or in Intact Cellular Systems in Vitro.” Chemico-Biological Interactions 105 (1): 17–34.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Fennell, T. R., and C. D. Brown. 2001. “A Physiologically Based Pharmacokinetic Model for Ethylene Oxide in Mouse, Rat, and Human.” Toxicology and Applied Pharmacology 173 (3): 161–75.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Spahiu, Linda, Johan Ålander, Astrid Ottosson-Wadlund, Richard Svensson, Carina Lehmer, Richard N. Armstrong, and Ralf Morgenstern. 2017a. “Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation.” Biochemistry 56 (24): 3089–98.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px"><span style="color:#000000">Csanády, G. A., P. E. Kreuzer, C. Baur, and J. G. Filser. 1996. “A Physiological Toxicokinetic Model for 1,3-Butadiene in Rodents and Man: Blood Concentrations of 1,3-Butadiene, Its Metabolically Formed Epoxides, and of Haemoglobin Adducts--Relevance of Glutathione Depletion.” Toxicology 113 (1-3): 300–305.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Morgenstern, R., R. Svensson, B. A. Bernat, and R. N. Armstrong. 2001. “Kinetic Analysis of the Slow Ionization of Glutathione by Microsomal Glutathione Transferase MGST1.” Biochemistry 40 (11): 3378–84.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Mayer, Robert J., and Armin R. Ofial. 2019. “Nucleophilicity of Glutathione: A Link to Michael Acceptor Reactivities.” Angewandte Chemie 58 (49): 17704–8.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">McMullin, Tami, Jill Brzezicki, Brian Cranmer, John Tessari, and Melvin Andersen. 2003. “Pharmacokinetic Modeling of Disposition and Time-Course Studies With [ 14 C]Atrazine.” Journal of Toxicology and Environmental Health. Part A 66 (10): 941–64.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Geenen, Suzanne, James W. T. Yates, J. Gerry Kenna, Frederic Y. Bois, Ian D. Wilson, and Hans V. Westerhoff. 2013. “Multiscale Modelling Approach Combining a Kinetic Model of Glutathione Metabolism with PBPK Models of Paracetamol and the Potential Glutathione-Depletion Biomarkers Ophthalmic Acid and 5-Oxoproline in Humans and Rats.” Integrative Biology: Quantitative Biosciences from Nano to Macro 5 (6): 877–88.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Hughes, Tyler B., Grover P. Miller, and S. Joshua Swamidass. 2015. “Site of Reactivity Models Predict Molecular Reactivity of Diverse Chemicals with Glutathione.” Chemical Research in Toxicology 28 (4): 797–809.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">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></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Singh, Mohan, Rajat Sandhir, and Ravi Kiran. 2010. “Oxidative Stress Induced by Atrazine in Rat Erythrocytes: Mitigating Effect of Vitamin E.” Toxicology Mechanisms and Methods 20 (3): 119–26.</span></span></p>
<p>Considering the empirical domain of the evidence, the GSH depletion leading to increased, reactive oxygen species is known to occur in fish and mammals, but, based on scientific reasoning, the biologically plausible domain of applicability can be eukaryotic organisms in general. It can be measured at any stage of life and in both male and female species.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-family:Arial,Helvetica,sans-serif">Biological plausibility for GSH depletion leading to ROS increase is rooted in the fact that this antioxidant is crucial to eliminate these reactive molecules from cells. When GSH is depleted from cytosol and mitochondria, there is an exaggerated accumulation of ROS, produced, mainly, by electron transport chain.</span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-family:Arial,Helvetica,sans-serif">Empirical evidence shows that this KER is commonly registered in several animal models, including vertebrates, and this is because it is conserved among taxa. Additionally, as expected, in vitro and in vivo data gathered for the three chosen compounds highlight that.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">(Tirmenstein et al. 2000), analyzing the relation between GSH depletion and ROS production in rat hepatocyte suspensions exposed to 5 mM DEM, for a period of 4 h, noted that GSH is used up, leading to overproduction and hyperaccumulation of ROS in mitochondria. </span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Still in the same work, Tirmenstein et al. (2000) showed that at lower concentrations, DEM, an alkylating agent, does not interfere with ROS production, but it exhausts GSH at different levels, pointing up that decrease in GSH content is affected for that stressor at concentrations equal or lower to those that induce a rise in ROS levels. DEM at 0.1, 0.5, 1, 2.5 and 5 mM for five hours caused GSH depletion in hepatocytes at all concentrations in a dose-dependent manner. However, only 5 mM of the compound was able to consume GSH to the point that this antioxidant was kept below detection levels (4%) and led to overproduction of ROS.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">In relation to ATZ, in PC12 cells (rat pheochromocytoma cell line), at 232 μM, the herbicide causes a decrease in GSH content followed by a rise in ROS levels after 24 h of exposure (Abarikwu et al. 2011). In in vivo models, ATZ-treated rat erythrocytes (300 mg/Kg body weight, daily) for 7, 14 and 21 days, displayed significant GSH consumption with concomitant increase in superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities, which suggests a rise in ROS levels. And in BALB/c mice, ATZ doses (100, 200, or 400 mg/Kg body weight/daily) administered for 21 days led to a reduction in GSH content and increase of ROS levels in splenocytes in a dose-dependent manner (Gao et al. 2016). </span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">These data are in accordance with two other studies carried out with other experimental models. Human neuroblastoma SH-SY5Y cells exposed to ATZ (0.3 mM) for 24 h displayed both a drop in GSH content as well as ROS overproduction (Abarikwu et al. 2011). Additionally, in zebrafish embryos exposed to atrazine at 0.1 mM for 96 h exhibited a decrease in GSH content followed by a rise in CAT enzyme activity, responsible for H2O2 scavenging, suggesting in this model that decrease in GSH content induces a rise in ROS levels (Adeyemi, da Cunha Martins-Junior, and Barbosa 2015). </span></p>
<p><br />
<span style="font-family:Arial,Helvetica,sans-serif">This same response pattern is observed using Hg as a stressor. Cultured human bronchial epithelial cells (BEAS-2B cell line) exposed to mercury (II) chloride (2, 4, 6, and 8 ppm) for 24 h, and monitored every 3 h in order to measure GSH levels, displayed a decrease in GSH content at all concentrations from the third hour in a dose-dependent manner. A 60% drop in GSH content was kept constant during all exposure time at 8 ppm, whereas all other concentrations induced a constant diminishment of GSH content from 12 to 24 h post-exposure, but not that high. Likewise, a dose-dependent pattern of ROS generation was observed in BEAS-2B cell line, but only after 24 h of exposure. The authors still exposed these cells to 8 ppm of mercury (for 3, 6, 12 and 24 h) and noted a quick increase in ROS levels from 12 h of exposure on, but only after 24 h it was observed a noticeable increase in ROS levels – 3 times greater than the control group (Park and Park 2007). </span></p>
<p><br />
<span style="font-family:Arial,Helvetica,sans-serif">The same response pattern in in vitro and in vivo models is found if GSH depletion is specifically stimulated for the inhibition of its de novo synthesis, revealing the direct causality among KEs. In HT-22 mouse hippocampal cell line submitted to 50 µM buthionine sulfoximine (BSO), a traditional GSH synthesis inhibitor, leads to glutathione depletion so that an initial increase in ROS levels takes place afterwards (Tan et al. 1998). (Armstrong et al. 2002) tracking GSH and ROS levels in human B lymphoma cell lines (PW) submitted to 1 mM BSO, for a long period of time (24, 48 and 72 h) concluded that GSH depletion is directly responsible for the increase of ROS levels and a drop in mitochondrial GSH content is a key factor for the exponential augmentation of these free radicals. </span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Corroborating these data, adult rats treated with BSO 20 and 30 mM, for 10 days, diligently, showed a reduction of, respectively, 44.25 % and 60.14 % of liver GSH content, while H2O2 levels underwent an augmentation of 42 and 60%, in that order (Ford et al. 2006).</span></p>
<p><br />
<span style="font-family:Arial,Helvetica,sans-serif">Thus, from this overview of experimental data, it is noted that GSH depletion needs to happen previously in the course of time so that ROS production is triggered in cells and tissues and, besides that, the greater the depletion, the more pronounced the increase in ROS levels. In addition, this assessment reveals that upstream KE is affected by stressor in doses equal or lower to those that unleash downstream KE, as well as the upstream KE is more frequent than the downstream one in equivalent stress degrees. In addition, this provides robustness to dose, time and incidence concordances for this KER. Just as important, the relation is also quite conserved through several taxa (Trachootham et al. 2008).</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-family:Arial,Helvetica,sans-serif">The close relation between GSH depletion and increase in ROS levels is a well-established biological process, which is a result of diverse experimental evidence.</span></p>
<p> </p>
<strong>Response-response relationship</strong>
<p><span style="font-family:Arial,Helvetica,sans-serif">Drop in GSH levels and increase in ROS generation changes cellular redox potential, which can be calculated by the Nernst equation (Han et al. 2006): </span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">where E<sub>cell</sub> is cell electrochemical voltage, E<sup>o</sup> is the electromotive force, R is molar gas constant, T is the temperature in Kelvin, F is the Faraday constant, n is the number of electrons transferred in the reaction, and Q is [GSH]2/[GSSG].</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">If GSH levels drop until a certain threshold (~30 - 40% of depletion) in mitochondria, there is an excessive H<sub>2</sub>O<sub>2</sub> release in cells (Han et al. 2006) and, hence, ROS exacerbation.</span></p>
<strong>Time-scale</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">For HT22 cells exposed to 50 µM BSO (for 10 h), ROS production occurs in two phases: an initial slow increase for the first 6 h, followed by a much higher rate. The latter high rate of increase in ROS only starts after the cellular GSH levels drop to nearly zero (Tan et al. 1998).</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Moreover, isolated rat hepatocyte suspensions exposed to DEM (0.5, 1, 2.5 and 5 mM) for 5 h reach maximum levels of GSH depletion after 1 h of exposure (Tirmenstein et al. 2000), whereas the maximum increase in ROS levels is observed only after four hours at the two highest concentrations of each depleter.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">GSH has its levels reduced by more than 95% in PW cells after around 8 h of exposure to BSO and reacher maximum depletion level at 48 h, when mitochondrial GSH supplies become undetectable as well, whereas ROS levels undergo a slight increase only 24 h post-exposure and reaches maximum values after 60 h of treatment (Armstrong et al. 2002).</span></p>
<td><span style="font-family:Arial,Helvetica,sans-serif">restores the activity of antioxidant enzymes</span></td>
<td><span style="font-family:Arial,Helvetica,sans-serif">Singh, Sandhir, and Kiran 2010</span></td>
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</tbody>
</table>
</div>
<h4>References</h4>
<p><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><span style="font-family:Arial,Helvetica,sans-serif">Abarikwu, Sunny O., Ebenezer O. Farombi, Mahendra P. Kashyap, and Aditya B. Pant. 2011. “Kolaviron Protects Apoptotic Cell Death in PC12 Cells Exposed to Atrazine.” Free Radical Research 45 (9): 1061–73.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Gao, Shuying, Zhichun Wang, Chonghua Zhang, Liming Jia, and Yang Zhang. 2016. “Oral Exposure to Atrazine Induces Oxidative Stress and Calcium Homeostasis Disruption in Spleen of Mice.” Oxidative Medicine and Cellular Longevity 2016 (November): 7978219.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif; font-size:1rem">Adeyemi, Joseph A., Airton da Cunha Martins-Junior, and Fernando Barbosa Jr. 2015. “Teratogenicity, Genotoxicity and Oxidative Stress in Zebrafish Embryos (Danio Rerio) Co-Exposed to Arsenic and Atrazine.” Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP 172-173 (April): 7–12.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Park, Eun-Jung, and Kwangsik Park. 2007. “Induction of Reactive Oxygen Species and Apoptosis in BEAS-2B Cells by Mercuric Chloride.” Toxicology in Vitro: An International Journal Published in Association with BIBRA 21 (5): 789–94.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Tan, S., Y. Sagara, Y. Liu, P. Maher, and D. Schubert. 1998. “The Regulation of Reactive Oxygen Species Production during Programmed Cell Death.” The Journal of Cell Biology 141 (6): 1423–32.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Armstrong, J. S., K. K. Steinauer, B. Hornung, J. M. Irish, P. Lecane, G. W. Birrell, D. M. Peehl, and S. J. Knox. 2002. “Role of Glutathione Depletion and Reactive Oxygen Species Generation in Apoptotic Signaling in a Human B Lymphoma Cell Line.” Cell Death & Differentiation. https://doi.org/10.1038/sj.cdd.4400959.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Ford, Rebecca J., Drew A. Graham, Steven G. Denniss, Joe Quadrilatero, and James W. E. Rush. 2006. “Glutathione Depletion in Vivo Enhances Contraction and Attenuates Endothelium-Dependent Relaxation of Isolated Rat Aorta.” Free Radical Biology & Medicine 40 (4): 670–78.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Trachootham, Dunyaporn, Weiqin Lu, Marcia A. Ogasawara, Rivera-Del Valle Nilsa, and Peng Huang. 2008. “Redox Regulation of Cell Survival.” Antioxidants & Redox Signaling 10 (8): 1343–74.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Han, Derick, Naoko Hanawa, Behnam Saberi, and Neil Kaplowitz. 2006. “Mechanisms of Liver Injury. III. Role of Glutathione Redox Status in Liver Injury.” American Journal of Physiology. Gastrointestinal and Liver Physiology 291 (1): G1–7.</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Singh, Mohan, Rajat Sandhir, and Ravi Kiran. 2010. “Oxidative Stress Induced by Atrazine in Rat Erythrocytes: Mitigating Effect of Vitamin E.” Toxicology Mechanisms and Methods 20 (3): 119–26.</span></p>
<p> </p>
</div>
<div>
<h4><a href="/relationships/2460">Relationship: 2460: Increased, Reactive oxygen species leads to Increase, LPO</a></h4>
<h4><a href="/relationships/2460">Relationship: 2460: Increase, ROS leads to Increase, LPO</a></h4>
<p>Considering the empirical domain of the evidence, the increased, reactive oxygen species leading to increased, lipid peroxidation is known to occur in fish and mammals, but, based on scientific reasoning, the biologically plausible domain of applicability can be eukaryotic organisms in general. It can be measured at any stage of life and in both male and female species.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Biological plausibility of this KER lies in the fact that reactive species, in excess, react and change macromolecules such as proteins, nucleic acids and lipids. Membrane lipids are particularly susceptible to damage by free radicals, as they are composed by unsaturated fatty acids (Su et al. 2019). Hence, increase in ROS production beyond antioxidant system defense capability of cells enables free circulation of molecules such as O2·−, HO·, H2O2, which removes electrons from membrane lipids and then triggers lipid peroxidation (Auten and Davis 2009; Su et al. 2019). </span></p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Analyses performed to support this relation show that KER3 is unchained by the three previously selected xenobiotics, as well as it takes place in a conserved way among species. Connection among the KEs is observed in both in vitro experimental models and in vivo systems, including fishes, birds and mammals.</span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">In cultures of rat hepatocytes, progressive ROS increase during 4 hours of treatment, triggered by DEM (5 mM), is followed by a continuous growth in levels of thiobarbituric acid reactive substances (TBARS), lipid peroxidation markers (Tirmenstein et al. 2000). This chemical depletes GSH content, leading to an augmentation of ROS levels and, consequently, to lipid peroxidation. In an in vivo model, 52 μM of DEM intraperitoneally injected in male Balb/c mice for two weeks caused a significant decrease in the GSH, increase in GSSG, ROS generation and increase in lipid peroxidation in testicles (Kalia and Bansal 2008).</span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">ATZ (46.4 µM) causes an increase of 48.97% of ROS and of 12.5% in MDA content in cultures of Sertoli-Germ cells from Wistar rats (25–28 days old), after, respectively, 3 and 24 h post-exposure. At a higher concentration (232 µM), these cells reach a maximum peak of ROS production after 6h of exposure, while MDA generation gets to the peak only after 24 h of treatment (Abarikwu, Pant, and Farombi 2012). In in vivo model, ATZ (38.5, 77 e 154 mg/Kg bw/day) led to a decrease in total antioxidant capacity (TAC) in a dose-dependent manner in male Sprague-Dawley rats of Specific Pathogen Free (SPF) ATZ-treated for 30 days. Which indirectly suggests increase in ROS levels – and increased malondialdehyde (MDA) content in 154 mg/Kg (Song et al. 2014). </span></p>
<p style="text-align:justify"><br />
<span style="font-family:Arial,Helvetica,sans-serif">In relation to Hg, it was found that male young Wistar rats exposed to an initial dose of 4.6 μg/Kg of this metal (with following doses of 0.07 μg/Kg/day) displayed an increase in ROS levels, followed by an elevation of MDA content in testicles and epididymis of these rats 60 days post-exposure (Rizzetti et al. 2017). Other assays still carried out with male rats showed that the heavy metal induces oxidative stress with a single subcutaneous dose of 5 mg/Kg, by a substantial diminishment of activity of the main testicle antioxidant enzymes: SOD, CAT and GPX. Consequently, blood hydroperoxide and testicle MDA levels rose in a relevant way (El-Desoky et al. 2013).</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Furthermore, Hy-Line Brown laying hens fed with 4 experimental diets containing graded levels of Hg at 0.280, 3.325, 9.415, and 27.240 mg/Kg, respectively, for 10 weeks had GSH content significantly decreased in all Hg-treatment groups in ovaries, whilst SOD, CAT, GPX and glutathione reductase (GR) enzyme activities were significantly reduced, pointing to ROS accumulation. MDA content strongly increased in the 27.240-mg/Kg Hg group (Ma et al. 2018). </span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Hence, it can be deduced that, as in other adjacent relations evaluated, there is also evidence here that upstream KE is initially required in order to downstream KE take place, which reaffirms time concordance. Besides this, data enhance dose and incidence concordances for this KER.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Mechanisms involving lipid peroxidation, such as that one caused by ROS accumulation in cells, have been investigated for decades (Tirmenstein et al. 2000; Yin, Xu, and Porter 2011; Su et al. 2019). For this reason, there is much experimental data about response-response relationships or a growth of upstream KE in relation to downstream KE.</span></p>
<strong>Response-response relationship</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">This mechanism can be better understood through a process chain that consists of initiation, propagation and termination, as discussed by (Yin, Xu, and Porter 2011). In their review, these authors summarized a series of chemical reactions that develop during all this self-oxidation process and represent them in a schematic manner, as displayed in figure below.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Furthermore, although phospholipid oxidizability is lower, once their rate of diffusion in membranes is slower, the kinetics for this kind of reaction shown in figure follows the same law of velocity (steady-state rate) of homogeneous systems (equation below) (Yin, Xu, and Porter 2011). Oxygen consumption of the equation represents the rate of steady state, while rate of radical generation is defined by R<sub>i</sub>, the constant of propagation rate is expressed as k<sub>p</sub> and the termination rate constant for the reaction is called k<sub>t</sub>.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">For instance, empirical evidences show that rat hepatocytes begin ROS production after the first 30 minutes of DEM exposition (5 mM), growing linearly for all the remaining time, whereas the increase in products of lipid peroxidation (TBARS) starts only from the first hour of exposure (Tirmenstein et al. 2000).</span></p>
<td><span style="font-family:Arial,Helvetica,sans-serif">Auten and Davis 2009</span></td>
</tr>
</tbody>
</table>
</div>
<h4>References</h4>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Su, Lian-Jiu, Jia-Hao Zhang, Hernando Gomez, Raghavan Murugan, Xing Hong, Dongxue Xu, Fan Jiang, and Zhi-Yong Peng. 2019. “Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis.” Oxidative Medicine and Cellular Longevity 2019 (October): 5080843.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Auten, Richard L., and Jonathan M. Davis. 2009. “Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details.” Pediatric Research 66 (2): 121–27.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Tirmenstein, M. A., F. A. Nicholls-Grzemski, J. G. Zhang, and M. W. Fariss. 2000. “Glutathione Depletion and the Production of Reactive Oxygen Species in Isolated Hepatocyte Suspensions.” Chemico-Biological Interactions 127 (3): 201–17.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Kalia, Sumiti, and M. P. Bansal. 2008. “Diethyl Maleate-Induced Oxidative Stress Leads to Testicular Germ Cell Apoptosis Involving Bax and Bcl-2.” Journal of Biochemical and Molecular Toxicology 22 (6): 371–81.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Abarikwu, S. O., E. O. Farombi, and A. B. Pant. 2011. “Biflavanone-Kolaviron Protects Human Dopaminergic SH-SY5Y Cells against Atrazine Induced Toxic Insult.” Toxicology in Vitro: An International Journal Published in Association with BIBRA 25 (4): 848–58.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Rizzetti, Danize Aparecida, Caroline Silveira Martinez, Alyne Goulart Escobar, Taiz Martins da Silva, José Antonio Uranga-Ocio, Franck Maciel Peçanha, Dalton Valentim Vassallo, Marta Miguel Castro, and Giulia Alessandra Wiggers. 2017. “Egg White-Derived Peptides Prevent Male Reproductive Dysfunction Induced by Mercury in Rats.” Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 100 (February): 253–64.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">El-Desoky, Gaber E., Samir A. Bashandy, Ibrahim M. Alhazza, Zeid A. Al-Othman, Mourad A. M. Aboul-Soud, and Kareem Yusuf. 2013. “Improvement of Mercuric Chloride-Induced Testis Injuries and Sperm Quality Deteriorations by Spirulina Platensis in Rats.” PloS One 8 (3): e59177.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Ma, Yan, Mingkun Zhu, Liping Miao, Xiaoyun Zhang, Xinyang Dong, and Xiaoting Zou. 2018. “Mercuric Chloride Induced Ovarian Oxidative Stress by Suppressing Nrf2-Keap1 Signal Pathway and Its Downstream Genes in Laying Hens.” Biological Trace Element Research 185 (1): 185–96.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Yin, Huiyong, Libin Xu, and Ned A. Porter. 2011. “Free Radical Lipid Peroxidation: Mechanisms and Analysis.” Chemical Reviews 111 (10): 5944–72.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Auten, Richard L., and Jonathan M. Davis. 2009. “Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details.” Pediatric Research 66 (2): 121–27.</span></p>
<p> </p>
</div>
<div>
<h4><a href="/relationships/2879">Relationship: 2879: Increase, LPO leads to impaired, Fertility</a></h4>
<h4><a href="/relationships/2879">Relationship: 2879: Increase, LPO leads to decreased, Fertility</a></h4>
<p>Considering the empirical domain of the evidence, the increased lipid peroxidation leading to impaired fertility is known to occur in fish and mammals, but, based on scientific reasoning, the biologically plausible domain of applicability may also have relevance for amphibians, reptiles, birds and and invertebrates with sexual reproduction. It can be measured in juveniles and adults and in both male and female species.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Biological plausibility of this KER lies in the fact that lipid peroxidation in gonad membranes induces morphological changes in seminiferous tubules, and degeneration of ovarian follicles and Sertoli and Leydig cells in testicles, damage to gametic cells, and, consequently, reduction of their viability. This directly affects animal reproductive capability, for it reduces quality and production of oocytes and spermatocytes, as well as decreases egg and sperm release (spawn), leading to a drop-in fertilization rate (Tillitt et al. 2010; Papoulias et al. 2014; Song et al. 2014; Dasmahapatra et al. 2020; Biswas et al. 2020; Mu et al. 2022). </span></p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Just like the other KERs, the adjacent relation here assessed is also observed in different species and models used in toxicological studies. Nevertheless, evidence gathered here shows that occurrence of lipid peroxidation triggered by ATZ, DEM and Hg leading to fertility impairment is limited to fishes and mammals. In this case, mammals have their reproductive capability reduced mainly because of morphological changes and poor quality of gametes.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Sexually mature female Wistar rats treated by a daily gavage of 0, 5, 25 and 125 mg/Kg ATZ for 28 consecutive days exhibited lipid peroxidation and ovarian atresia significantly increased in a dose-dependent manner in ATZ-treated animals (Zhao et al. 2014). In male Sprague-Dawley rats exposed to ATZ by gavage (0, 38.5, 77, and 154 mg/Kg bw/day) for 30 days had significant adverse effects on the reproductive system, with the animals showing a decreased level of total antioxidant capacity (TAC) in a dose-dependent manner, a depletion in GSH and an increased MDA content at the highest dose, followed by not only irregular and disordered arrangement of the seminiferous epithelium, but also a decreased number of spermatozoa and augmented spermatozoa abnormality rate in groups treated with 77, and 154 mg/Kg of ATZ (Song et al. 2014).</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Corroborating these data, Farombi et al. (2013) showed that male Wistar rats administered with ATZ at a dose equivalent to 120 mg/Kg body weight each day for 16 days displayed augmented MDA levels in testicles and epididymis, as well an increased number of sperm abnormalities and reduced sperm production, sperm motility and epididymal and testicular sperm numbers. Moreover, degeneration of seminiferous tubules in testicles with the presence of defoliation was noted as well (Farombi et al. 2013). In adult male Albino rats, ATZ (120 mg/Kg bw) causes significantly increased malondialdehyde (MDA) serum level and also diminished total antioxidant capacity (TAC), besides inducing a significant rise in sperm cell abnormalities (Abdel Aziz et al. 2018). In addition, pathological lesions such as disorganized seminiferous tubules with degenerated and irregularly arranged necrotized germinal cells were also reported (Abdel Aziz et al. 2018). This very ATZ dosage orally administered in rats caused an increase in MDA formation in the liver, testis and epididymis, along with an inhibition of GST activities, and decreased epididymal and testicular sperm number, sperm motility, daily sperm production and increased number of dead and abnormal sperm in animals (Adesiyan et al. 2011).</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Kalia & Bansal (2008) found in their study that male Balb/c mice treated with DEM (52 μM) underwent a decrease in GSH content, and increased ROS generation and lipid peroxidation in testicles, followed by augmented apoptosis in germ cells, as well as a significant reduction in the number of these cells (Kalia and Bansal 2008). A lower dose of this compound (8.7 μM) daily and intraperitoneally injected, for two weeks, resulted in depleted GSH and increase in testis GSSG levels. As a consequence sperm motility was decreased by 40%, and epididymal sperm count was significantly reduced in DEM-treated animals. Beyond that, fertility status was also affected by DEM exposure, with a 34% diminishment compared to the control group, and there was a reduction in litter size as well (Kaur, Kalia, and Bansal 2006).</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Using Hg in order to induce oxidative stress, these kinds of results also occur in different taxa. Male Wistar rats continuously exposed to 0, 50 and 100 ppm Hg for 90 days through oral administration in the drinking water displayed a significant increase in testicular MDA, along with specific alterations in the histoarchitecture of testis, including disintegration of germinal epithelium of seminiferous tubules, detachment and degenerative changes of lining cells, increased space between the seminiferous tubules and their lumen enlarged in a dose-dependent manner (Boujbiha et al. 2009). Interestingly, Hg-treated males were mated with normal cyclic females and they showed a decline in reproductive performance. In another study, the heavy metal led to an increase in ROS and MDA levels in testes and epididymis 60 days post-exposure in Wistar rats submitted to a first dose of 4.6 μg/Kg and subsequent doses of 0.07 μg/Kg/day, as well as decreased sperm number, increased sperm transit time in epididymis and impaired sperm morphology (Rizzetti et al. 2017). In fishes, sublethal doses of mercury (II) chloride (0.04 and 0.12 ppm) for 30 days caused a significant increase in testicle lipid peroxidation, DNA fragmentation, and a decrease in sperm count, activity and motility in relation to the control group in African sharptooth catfish (<em>Clarias gariepinus</em>) (Ibrahim, Banaee, and Sureda 2019). In addition to that, histopathological alterations in testis sections including rupture of interlobular connective tissue, lessening of spermatogonia, derangement of spermatogenesis and low spermatozoa counting were also observed (Ibrahim, Banaee, and Sureda 2019). </span><br />
</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">With regard to KER4, several studies have brought quantitative data concerning the negative correlation between lipid peroxidation and fertility disorders of vertebrate organisms (Gomez, Irvine, and Aitken 1998; Hsieh, Chang, and Lin 2006; Aitken et al. 2007; Abarikwu et al. 2010; Mihalas et al. 2017).</span></p>
<strong>Response-response relationship</strong>
<p><span style="font-family:Arial,Helvetica,sans-serif">According to (Gomez, Irvine, and Aitken 1998), there is a negative relationship between malondialdehyde and 4-hydroxyalkenal production (MDA + 4-HA) and loss of motility in human spermatozoa. The higher the amount of these peroxidation products, the lower the cell motility. A negative correlation between sperm numbers and testicular and epididymal MDA levels (-0.85 and -0.68 correlation coefficient r, respectively) was also found by (Abarikwu et al. 2010) in rats exposed to ATZ for 7 and 16 days. Conversely, the authors observed a positive correlation between abnormal sperm rate and testicular and epididymal MDA levels (+0.78 and +0.89). <br />
Hsieh et al. (2006), assessing MDA levels and sperm quality of 51 subfertile men, were able to establish two formulas to associate lipid peroxidation with sperm concentration and motility, which are represented, respectively, by: </span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">MDA = - 0.014 x sperm motility + 2.62. </span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">On the other hand, (Mihalas et al. 2017) brought important quantitative data about the direct relation between lipid peroxidation and reduction of quality in oocytes. Experimental evidences showed that the lipid peroxidation product 4-HNE, at 0, 5, 10, 20, 30 and 50 µM, induces a dose-dependent decrease in meiotic competence during in vitro oocyte maturation, as well as aneuploidies in germinal vesicle (GV) oocytes from 20 µM of 4-HNE. They still reported this happens because tubulins, component proteins of microtubules of the mitotic spindle, generate adducts with 4-HNE. </span></p>
<h4>References</h4>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Tillitt, Donald E., Diana M. Papoulias, Jeffrey J. Whyte, and Catherine A. Richter. 2010. “Atrazine Reduces Reproduction in Fathead Minnow (Pimephales Promelas).” Aquatic Toxicology 99 (2): 149–59.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Papoulias, Diana M., Donald E. Tillitt, Melaniya G. Talykina, Jeffrey J. Whyte, and Catherine A. Richter. 2014. “Atrazine Reduces Reproduction in Japanese Medaka (Oryzias Latipes).” Aquatic Toxicology 154 (September): 230–39.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Song, Yang, Zhen Chao Jia, Jin Yao Chen, Jun Xiang Hu, and Li Shi Zhang. 2014. “Toxic Effects of Atrazine on Reproductive System of Male Rats.” Biomedical and Environmental Sciences: BES 27 (4): 281–88.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Dasmahapatra, Asok K., Doris K. Powe, Thabitha P. S. Dasari, and Paul B. Tchounwou. 2020. “Assessment of Reproductive and Developmental Effects of Graphene Oxide on Japanese Medaka (Oryzias Latipes).” Chemosphere 259 (November): 127221.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Biswas, Subhasri, Soumyajyoti Ghosh, Anwesha Samanta, Sriparna Das, Urmi Mukherjee, and Sudipta Maitra. 2020. “Bisphenol A Impairs Reproductive Fitness in Zebrafish Ovary: Potential Involvement of Oxidative/nitrosative Stress, Inflammatory and Apoptotic Mediators.” Environmental Pollution 267 (December): 115692.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Mu, Xiyan, Suzhen Qi, Jia Liu, Hui Wang, Lilai Yuan, Le Qian, Tiejun Li, et al. 2022. “Environmental Level of Bisphenol F Induced Reproductive Toxicity toward Zebrafish.” The Science of the Total Environment 806 (Pt 1): 149992.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Zhao, Fan, Kun Li, Lijing Zhao, Jian Liu, Qi Suo, Jing Zhao, Hebin Wang, and Shuhua Zhao. 2014. “Effect of Nrf2 on Rat Ovarian Tissues against Atrazine-Induced Anti-Oxidative Response.” International Journal of Clinical and Experimental Pathology 7 (6): 2780–89.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Farombi, E. O., S. O. Abarikwu, A. C. Adesiyan, and T. O. Oyejola. 2013. “Quercetin Exacerbates the Effects of Subacute Treatment of Atrazine on Reproductive Tissue Antioxidant Defence System, Lipid Peroxidation and Sperm Quality in Rats.” Andrologia 45 (4): 256–65.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Abdel Aziz, Rabie L., Ahmed Abdel-Wahab, Fatma I. Abo El-Ela, Nour El-Houda Y. Hassan, El-Shaymaa El-Nahass, Marwa A. Ibrahim, and Abdel-Tawab A. Y. Khalil. 2018. “Dose- Dependent Ameliorative Effects of Quercetin and L-Carnitine against Atrazine- Induced Reproductive Toxicity in Adult Male Albino Rats.” Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie 102 (June): 855–64.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Adesiyan, Adebukola C., Titilola O. Oyejola, Sunny O. Abarikwu, Matthew O. Oyeyemi, and Ebenezer O. Farombi. 2011. “Selenium Provides Protection to the Liver but Not the Reproductive Organs in an Atrazine-Model of Experimental Toxicity.” Experimental and Toxicologic Pathology: Official Journal of the Gesellschaft Fur Toxikologische Pathologie 63 (3): 201–7.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Kalia, Sumiti, and M. P. Bansal. 2008. “Diethyl Maleate-Induced Oxidative Stress Leads to Testicular Germ Cell Apoptosis Involving Bax and Bcl-2.” Journal of Biochemical and Molecular Toxicology 22 (6): 371–81.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Kaur, Parminder, Sumiti Kalia, and Mohinder P. Bansal. 2006. “Effect of Diethyl Maleate Induced Oxidative Stress on Male Reproductive Activity in Mice: Redox Active Enzymes and Transcription Factors Expression.” Molecular and Cellular Biochemistry 291 (1-2): 55–61.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Boujbiha, Mohamed Ali, Khaled Hamden, Fadhel Guermazi, Ali Bouslama, Asma Omezzine, Abdelaziz Kammoun, and Abdelfattah El Feki. 2009. “Testicular Toxicity in Mercuric Chloride Treated Rats: Association with Oxidative Stress.” Reproductive Toxicology 28 (1): 81–89.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Rizzetti, Danize Aparecida, Caroline Silveira Martinez, Alyne Goulart Escobar, Taiz Martins da Silva, José Antonio Uranga-Ocio, Franck Maciel Peçanha, Dalton Valentim Vassallo, Marta Miguel Castro, and Giulia Alessandra Wiggers. 2017. “Egg White-Derived Peptides Prevent Male Reproductive Dysfunction Induced by Mercury in Rats.” Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 100 (February): 253–64.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Ibrahim, Ahmed Th A., Mahdi Banaee, and Antoni Sureda. 2019. “Selenium Protection against Mercury Toxicity on the Male Reproductive System of Clarias Gariepinus.” Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP 225 (November): 108583.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Gomez, E., D. S. Irvine, and R. J. Aitken. 1998. “Evaluation of a Spectrophotometric Assay for the Measurement of Malondialdehyde and 4-Hydroxyalkenals in Human Spermatozoa: Relationships with Semen Quality and Sperm Function.” International Journal of Andrology 21 (2): 81–94.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Hsieh, Yao-Yuan, Chi-Chen Chang, and Chich-Sheng Lin. 2006. “Seminal Malondialdehyde Concentration but Not Glutathione Peroxidase Activity Is Negatively Correlated with Seminal Concentration and Motility.” International Journal of Biological Sciences 2 (1): 23–29.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Aitken, R. John, Jordana K. Wingate, Geoffry N. De Iuliis, and Eileen A. McLaughlin. 2007. “Analysis of Lipid Peroxidation in Human Spermatozoa Using BODIPY C11.” Molecular Human Reproduction 13 (4): 203–11.</span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif">Mihalas, Bettina P., Geoffry N. De Iuliis, Kate A. Redgrove, Eileen A. McLaughlin, and Brett Nixon. 2017. “The Lipid Peroxidation Product 4-Hydroxynonenal Contributes to Oxidative Stress-Mediated Deterioration of the Ageing Oocyte.” Scientific Reports 7 (1): 6247.</span></p>
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