Upstream eventActivation of Cyp2E1 in the liver
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
Cyp2E1 activation has two major outcomes: (1) the production of reactive, electrophilic metabolites, and (2) a significant increase in the half-life of the Cyp2E1 enzyme (Gonzalez 2007, Song, et al. 1989). The former is important because metabolites can go on to produce cellular damage by reacting with cellular nucleophiles. The latter is important because the Cyp2E1 catalytic cycle is prone to uncoupling (i.e., instead of incorporating an oxygen atom in to the substrate, the catalytic cycle is interrupted because a superoxide radical is released), which results in the release of reactive oxygen species (ROS) and an increase in cellular oxidative stress (Lieber 1999).
Weight of Evidence
It is well known that uncoupling of Cyp2E1 catalytic cycle results in the release of harmful reactive oxygen species in the cell (Lieber 1999).
Oxidative stress is produced during chronic activation (and uncoupling) of the Cyp2E1 catalytic cycle. The cytochrome P-450 catalytic cycle is known to undergo uncoupling leading to the production of ROS (Gorsky, et al. 1984, Loida and Sligar 1993, Meunier, et al. 2004). If this uncoupling occurs, a molecule of superoxide radical is released, which has the effect of interrupting the P450 catalytic cycle and releasing harmful ROS into the cell. Typically superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD), which is further reduced into the hydroxyl radical (OH•), and then to water. Other relevant cellular antioxidants include glutathione, thioredoxin, and peroxiredoxins. However, it is also possible for these ROS to scavenge electrons from cellular macromolecules (proteins, lipids, nucleic acids). Because Cyp2E1 is membrane-bound, ROS most commonly react with lipids and initiate lipid peroxidation. Further, Cyp2E1 can undergo NADPH-dependent ‘futile cycling’, which produces ROS and contributes to the occurrence of lipid peroxidation (Ekstrom and Ingelman-Sundberg 1989). Persistent oxidative stress also creates opportunities for oxidative damage to DNA, which is important because DNA damage is required for the initiation phase of carcinogenesis. The cellular sources and effects of ROS, as well as the corresponding enzymes and antioxidants are reviewed in:. (Nakazawa, et al. 1996).
Empirical Support for Linkage
Empirical data collected from different experiments strongly supports that oxidative stress and cytotoxicity are downstream of Cyp2E1 activation. Evidence of both temporal and dose-response concordance are available for a variety of chemical exposures.
Cyp2E1 protein levels increase when its substrate is present in a tissue. Therefore, prolonged exposure to substrate leads to prolonged activation of Cyp2E1, which is related to a substantial increase in cellular oxidative stress. For example, treatment with acetone or ethanol in male Sprague-Dawley rats results in an increase in Cyp2E1 protein levels in the liver. Increasing Cyp2E1 levels are linearly correlated to concomitant increases in NADPH oxidase, superoxide radical, hydrogen peroxide, and lipid peroxidation (TBARS). Both hydrogen peroxide production and lipid peroxidation are blocked in rat microsomes following inhibition of Cyp2E1 with anti-Cyp2E1 IgG (Ekstrom and Ingelman-Sundberg 1989). Ethanol treatment leads to correlated increases in both Cyp2E1 protein and lipid peroxidation in male Wister rats, C57BL/129SV mice, and superoxide dismutase (Sod) knockout mice (Kessova, et al. 2003, Nanji, et al. 1994). Wild type and humanized Cype2E1 knock-in mice have dose-dependent increases in Cyp2E1 protein and activity levels when exposed to ethanol, whereas Cyp2E1 knock-out mice do not. Further, the humanized mice show the largest increases in necrosis, inflammation, AST, ALT and TBARS, and the largest decrease in GSH levels of all three groups (Lu, et al. 2010). Exposure of male Sprague-Dawley rats to 95% oxygen results in a time-dependent increase in Cyp2E1 protein levels, superoxide radical production, and lipid peroxidation (TBARS) (various time-points over 78 hours). Lipid peroxidation is further increased following treatment of rat microsomes with carbon tetrachloride, or co-treatment of rats with oxygen, acetone and/or carbon tetrachloride (Tindberg and Ingelman-Sundberg 1989), established Cyp2E1 substrates. Cyp2E1 expressing HepG2 cells (called E47 cells) have higher baseline expression of anti-oxidant molecules thioredoxin and glutathione compared to non-Cyp2E1 expressing HepG2 cells (called C34 cells); they also have higher levels of ROS and lipid peroxidation (Yang, et al. 2011). Studies of ethanol- and Cyp2E1-dependent oxidative injury in HepG2 E47 and C34 cells, and in the liver, have been reviewed previously (Caro and Cederbaum 2004, Lu and Cederbaum 2008).
At the molecular level, global gene expression profiling of mice exposed to a carcinogenic dose of furan (a Cyp2E1 substrate) demonstrated that the most perturbed molecular pathway was the Nrf2 Oxidative Stress Response pathway (Jackson, et al. 2014).
Uncertainties or Inconsistencies
Quantitative Understanding of the Linkage
The quantitative relationships between the degree of Cyp2E1 activation required to lead to specific levels of oxidative stress have not been determined.
Evidence Supporting Taxonomic Applicability
Caro, A.A., Cederbaum, A.I., 2004. Oxidative stress, toxicology, and pharmacology of CYP2E1. Annu. Rev. Pharmacol. Toxicol. 44, 27-42.
Ekstrom, G., Ingelman-Sundberg, M., 1989. Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1). Biochem. Pharmacol. 38, 1313-1319.
Gonzalez, F.J., 2007. The 2006 Bernard B. Brodie Award Lecture. Cyp2e1. Drug metabolism and disposition: the biological fate of chemicals 35, 1-8.
Gorsky, L.D., Koop, D.R., Coon, M.J., 1984. On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction. J. Biol. Chem. 259, 6812-6817.
Jackson, A.F., Williams, A., Recio, L., Waters, M.D., Lambert, I.B., Yauk, C.L., 2014. Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan. Toxicol. Appl. Pharmacol. 274, 63-77.
Kessova, I.G., Ho, Y.S., Thung, S., Cederbaum, A.I., 2003. Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase. Hepatology 38, 1136-1145.
Lieber, C.S., 1999. Microsomal ethanol-oxidizing system (MEOS): The first 30 years (1968- 1998) - A review. Alcohol. Clin. Exp. Res. 23, 991-1007.
Lu, Y., Cederbaum, A.I., 2008. CYP2E1 and oxidative liver injury by alcohol. Free Radical Biology and Medicine 44, 723-738.
Lu, Y., Wu, D., Wang, X., Ward, S.C., Cederbaum, A.I., 2010. Chronic alcohol-induced liver injury and oxidant stress are decreased in cytochrome P4502E1 knockout mice and restored in humanized cytochrome P4502E1 knock-in mice. Free Radic. Biol. Med. 49, 1406-1416.
Meunier, B., de Visser, S.P., Shaik, S., 2004. Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes. Chem. Rev. 104, 3947-3980.
Nakazawa, H., Genka, C., Fujishima, M., 1996. Pathological aspects of active oxygens/free radicals. Jpn. J. Physiol. 46, 15-32.
Nanji, A.A., Zhao, S., Sadrzadeh, S.M., Dannenberg, A.J., Tahan, S.R., Waxman, D.J., 1994. Markedly enhanced cytochrome P450 2E1 induction and lipid peroxidation is associated with severe liver injury in fish oil-ethanol-fed rats. Alcohol. Clin. Exp. Res. 18, 1280-1285.
Song, B.J., Veech, R.L., Park, S.S., Gelboin, H.V., Gonzalez, F.J., 1989. Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization. J. Biol. Chem. 264, 3568-3572.
Tindberg, N., Ingelman-Sundberg, M., 1989. Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung. Biochemistry 28, 4499-4504.
Yang, L., Wu, D., Wang, X., Cederbaum, A.I., 2011. Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells. Free Radic. Biol. Med. 51, 185-196.