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Depletion, GSH leads to Increased, Reactive oxygen species
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Glutathione conjugation leading to reproductive dysfunction via oxidative stress||adjacent||High||High||Leonardo Vieira (send email)||Under Development: Contributions and Comments Welcome|
Life Stage Applicability
Key Event Relationship Description
Evidence Collection Strategy
Evidence Supporting this KER
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.
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.
(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.
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.
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).
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).
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).
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.
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).
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).
Uncertainties and Inconsistencies
Known modulating factors
Quantitative Understanding of the Linkage
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.
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):
where Ecell is cell electrochemical voltage, Eo 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].
If GSH levels drop until a certain threshold (~30 - 40% of depletion) in mitochondria, there is an excessive H2O2 release in cells (Han et al. 2006) and, hence, ROS exacerbation.
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).
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.
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).
Known Feedforward/Feedback loops influencing this KER
In relation to factors that can modulate and change KER2 response-response pattern, vitamin E is able to restore activity of antioxidant enzymes in rat erythrocytes, such as superoxide dismutase, catalase, and glutathione peroxidase, responsible for scavenging ROS (Singh, Sandhir, and Kiran 2010), suggesting the possibility of reestablishment of basal cell redox potential.
Domain of Applicability
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.