Upstream eventOxidative Stress
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Directness||Weight of Evidence||Quantitative Understanding|
|Chronic Cyp2E1 Activation Leading to Liver Cancer||directly leads to||Strong||Not Specified|
Life Stage Applicability
How Does This Key Event Relationship Work
Oxidative stress leads directly to cytotoxicity through lipid peroxidation. Lipid peroxidation occurs when ROS scavenge electrons from poly-unsaturated fatty acids (PUFA), including membrane phospholipids. Lipid peroxidation occurs in three steps: initiation (in which the PUFA radical is produced), propagation (in which PUFA radicals react with molecular oxygen and a non-radical molecule to produce a lipid peroxide and lipid radical), and termination (in which two radicals combine to form a non-radical). Left unchecked, the propagation chain reaction is highly damaging to cellular membranes. Lipid peroxidation of mitochondrial membranes has been shown to result in both necrosis and apoptosis. The former occurs due to decreased mitochondrial membrane potential leading to decreased ATP production. The latter is a result of mitochondrial permeability transition (MPT). The mitochondrial permeability transition (MPT) is a process that can lead to necrosis or apoptosis. It is an important cell death mechanism because it is sensitive to redox. Accumulation of ROS and depletion of glutathione trigger the mitogen activated protein kinase (MAPK) cascade (ASK1-->MKK4-->JNK), which recruits Bax to the outer mitochondrial membrane (Youle and Strasser 2008). Bax triggers the opening of mitochondrial permeability transition pore (MTP), through which cytochrome c is released, which triggers the caspase cascade and apoptosis. Alternatively, when the MTP opens across the inner and outer mitochondrial membranes, mitochondrial swelling and decoupling of oxidative phosphorylation (i.e., loss of ATP generation) leads to cell death by necrosis (Pessayre, et al. 2010, Rasola and Bernardi 2007).
Oxidative stress triggers cytotoxicity indirectly by modifying redox sensitive cellular molecules. Proteins with neighboring cysteine residues sense ROS through the oxidation of adjacent thiol groups (2SH, reduced; S=S, oxidized). Examples of this include: (1) the cellular anti-oxidant glutathione (GSH), which acts to ‘mop up’ ROS (GSH oxidized to GS=SG), and its depletion is associated with elevated cytotoxicity; (2) the cellular anti-oxidant thioredoxin, which inhibits the apoptosis signaling kinase 1 (Ask1) in its reduced form, but not in its oxidized form (Liu, et al. 2000, Saitoh, et al. 1998); and, (3) the mitochondrial permeability transition pore, which opens when oxidized (Petronilli, et al. 1994). Oxidative stress can also produce cell death through the production of oxidative damage to DNA. Examples of types of oxidative DNA damage include: (Sharma, et al. 2012, Shukla, et al. 2013, Skipper, et al. 2016).
Weight of Evidence
Strong. It is well known that cellular oxidative damage, especially by lipid peroxidation, is cytotoxic.
Empirical Support for Linkage
Strong. This is an extremely data-rich KER. A large number of studies have taken measures of both oxidative stress and cytotoxicity within the same study. Below, we summarize a few examples, in vitro and in vivo, to demonstrate some empirical data that provides strong support for this KER.
For example, exposure of HepG2 cells to the pesticide malathion demonstrates a relationship between ROS and cytotoxicity at higher levels of exposure (Moore, et al. 2010). After 48 h of exposure to 0, 6, 12, 18, and 24 mM malathion, lipid peroxidation increases from the first concentration, whereas cytoxicity (MTT assay) does not significantly increase until 18 and 24 mM.
Similarly, treatment of cultured premonocytic U937 cells with increasing concentrations of lipid peroxidation-inducing agents (tert-butylhydroperoxide and 2,2’-azobis (2-amindinopropane) hydrochloride) results in increased lipid peroxidation within 30 min, and subsequent declines in relative survival (trypan blue assay) six hours post-exposure (Park, et al. 2002). In parallel, the major anti-oxidant enzymes catalase, SOD, G6PD, and GPx are deactivated in a dose-dependent fashion that is concordant with declines in relative survival.
Studies on primary rat hepatocytes show that a dose-dependent increase in TBARS is associated with a concomitant increase in cytotoxicity following exposure to fumonisin B1 (FB1) (Abel and Gelderblom 1998). Moreover, addition of the antioxidant alpha-tocopherol significantly decreases cytotoxicity and decreases TBARS to basal levels, supporting that lipid peroxidation contributes to the cytotoxic effects of FB1.
In female Wistar rat hepatocyte cultures, exposure to chloroform causes a small dose-dependent increase in M(1)dG adducts (a marker of lipid perodixation; occurs at 4 mM and above), DNA strand breakage (8 mM and above) and lipid peroxidation (at 4 mM and above). GSH depletion occurs in association with cytotoxicity (20 mM; lactate dehydrogenase release). Carbon tetrachloride (1 and 4 mM) exposure produces a small elevation in M(1)dG adducts and increases in 8-oxodG occur at the threshold of, and concomitant with, cytotoxicity (4 mM). (Beddowes, et al. 2003).
Similarly, exposure of 12-week old male Long Evans rats to increasing doses of cadmium (Cd) (i.p. injection) reveals that lipid peroxidation (TBARS) occurs in liver at low to medium doses, below those inducing tissue necrosis (Manca, et al. 1991). In this study, liver injury occurred above 125 ug Cd/kg (increased serum ALT and SDH). Levels of TBARS were similar in both the 125 and the 250 ug/doses, though only reached statistical significance relative to controls at 250 ug/kg in the liver. However, subsequent time-series analysis revealed increased liver TBARS after 2 h exposure to both 25 and 500 ug Cd/kg, prior to any changes in serum ALT, SDH, and tissue ALP (at the low dose, ALP was the only marker that was slightly increased at 6 hour). Therefore, lipid peroxidation precedes liver damage in this study.
Uncertainties or Inconsistencies
There exist some examples where measures of cytotoxicity could be observed below doses where assays for endpoints of oxidative stress were measured. However, it is difficult to compare endpoints measured using assays with different specificities and sensitivities. Quite generally, there is a high degree of association between measures of oxidative stress and cytotoxicity across tissues and species.
Quantitative Understanding of the Linkage
Unable to determine.
Evidence Supporting Taxonomic Applicability
Abel, S., Gelderblom, W.C., 1998. Oxidative damage and fumonisin B1-induced toxicity in primary rat hepatocytes and rat liver in vivo. Toxicology 131, 121-131.
Beddowes, E.J., Faux, S.P., Chipman, J.K., 2003. Chloroform, carbon tetrachloride and glutathione depletion induce secondary genotoxicity in liver cells via oxidative stress. Toxicology 187, 101-115.
Liu, H., Nishitoh, H., Ichijo, H., Kyriakis, J.M., 2000. Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin. Mol. Cell. Biol. 20, 2198-2208.
Manca, D., Ricard, A.C., Trottier, B., Chevalier, G., 1991. Studies on lipid peroxidation in rat tissues following administration of low and moderate doses of cadmium chloride. Toxicology 67, 303-323.
Moore, P.D., Yedjou, C.G., Tchounwou, P.B., 2010. Malathion-induced oxidative stress, cytotoxicity, and genotoxicity in human liver carcinoma (HepG2) cells. Environ. Toxicol. 25, 221-226.
Park, J.E., Yang, J.H., Yoon, S.J., Lee, J.H., Yang, E.S., Park, J.W., 2002. Lipid peroxidation-mediated cytotoxicity and DNA damage in U937 cells. Biochimie 84, 1199-1205.
Pessayre, D., Mansouri, A., Berson, A., Fromenty, B., 2010. Mitochondrial involvement in drug-induced liver injury. Handb. Exp. Pharmacol. (196):311-65. doi, 311-365.
Rasola, A., Bernardi, P., 2007. The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis. Apoptosis 12, 815-833.
Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., Ichijo, H., 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO Journal 17, 2596-2606.
Sharma, V., Singh, P., Pandey, A.K., Dhawan, A., 2012. Induction of oxidative stress, DNA damage and apoptosis in mouse liver after sub-acute oral exposure to zinc oxide nanoparticles. Mutat. Res. 745, 84-91.
Shukla, R.K., Kumar, A., Gurbani, D., Pandey, A.K., Singh, S., Dhawan, A., 2013. TiO(2) nanoparticles induce oxidative DNA damage and apoptosis in human liver cells. Nanotoxicology 7, 48-60.
Skipper, A., Sims, J.N., Yedjou, C.G., Tchounwou, P.B., 2016. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress. Int. J. Environ. Res. Public. Health. 13, 10.3390/ijerph13010088.
Youle, R.J., Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47-59.