Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Cyp2E1 Activation Leading to Liver Cancer||MolecularInitiatingEvent|
Key Event Description
Cyp2E1 is a membrane-bound monooxygenase that is primarily located in zone 3 hepatocytes (Ingelman-Sundberg, et al. 1988, Tsutsumi, et al. 1989). Although it is also expressed in other tissues (http://www.genecards.org/cgi-bin/carddisp.pl?gene=CYP2E1), the body of literature on CYP2E1 is focussed on measurement in liver. CYP2E1 is primarily located in the endoplasmic reticulum, but can also be present in the mitochondria. It is a phase I metabolism enzyme that catalyzes the oxidation of low molecular weight substrates. Unlike most cytochrome P450 enzymes, Cyp2E1 is constitutively expressed (i.e., its expression is not transcriptionally controlled by substrate-bound nuclear receptors). Alternatively, exposure to a substrate increases its activity through post-translational stabilization of the molecule. Thus, the presence of substrate significantly increases the half-life of the Cyp2E1 enzyme thereby allowing it to be active for a longer period of time (Gonzalez 2007, Song, et al. 1989). The sustained activation of Cyp2E1 due to the presence of the chemical substrate is required for this MIE to produce downstream adverse effects.
Cyp2E1 is also regulated by the ubiquitin-proteasome pathway and the involvement of hsp-based chaperone (Morishima et al. 2005); however, this mechanism of regulation is not discussed further herein.
How It Is Measured or Detected
- Mixed function oxidase catalytic activity. These assays have been thoroughly reviewed by Cederbaum (2014). The paper describes preparation of microsomes from both liver homogenates and cell cultures for testing Cyp2E1 activity. Briefly, the ratio of 6-hydroxychlorzoxazone/chlorzoxazone can be used to estimate levels of CYP2E1 in humans (Girre, et al. 1994). In addition, the oxidation of para-nitrophenol (PNP) to para nitrocatechol is an efficient and relatively specific assay to determine catalytic activity dependent on CYP2E1 [e.g., (Koop 1986, Koop, et al. 1989, Reinke and Moyer 1985)]. Other assays are described within the review article by Cederbaum.
- Western blot or Immunohistochemistry. Following chemical treatment, Cyp2E1 protein levels should increase if it is involved in the metabolism of that substrate. Western blot (of protein extracted from liver or cultured cells) or immunohistochemistry (of fixed liver or cultured cells) using anti-Cyp2E1 antibodies is the most straightforward approach for directly measuring increased levels of Cyp2E1.
- HepG2 cells. A compound’s Cyp2E1-dependence can be determined by comparing toxic effects in HepG2 versus HepG2-E47 cells. HepG2 cells are immortalized human hepatoma cells that do not express Cyp2E1; whereas, HepG2-E47 cells over-express Cyp2E1 (by recombinant retroviral infection). Chemicals that are metabolically activated by Cyp2E1 will cause cytotoxicity and oxidative stress in the E47 cells only. Toxicity can be blocked by treatment with antioxidants or Cyp2E1 inhibitors. Toxicity is exacerbated when glutathione is depleted (Wu and Cederbaum 2005) (e.g., ethanol (Cederbaum, et al. 2001, Chen and Cederbaum 1998, Chen, et al. 1998, Dai, et al. 1993).
- Measurement of chemical oxidation by Cyp2E1 in liver microsomes; described in the methodology review by Cederbaum (Cederbaum 2014). Reactions use specific probes to confirm that the compound undergoes oxidation, and that this oxidation reaction is catalyzed by Cyp2E1. See also: (Koop 1986, Koop, et al. 1989, Reinke and Moyer 1985).
- Cyp2E1 knock-out mouse. Chemical exposures in knockout mice are conducted and the production of the anticipated metabolites is measured. Lack of metabolite production indicates that Cyp2E1 is required for the chemical’s metabolism. Effects in knock-out mice are always measured in reference to wild-type (control) mice, which allows investigators to attribute the altered phenotype to gene product that has been knocked-out. Studies in Cyp2E1 knockout mice indicate the following chemicals interact with it: carbon tetrachloride (Wong, et al. 1998), acetone (Bondoc, et al. 1999), benzene (Powley and Carlson 2001), thioacetamide (Chilakapati, et al. 2007), trichloroethylene (Kim and Ghanayem 2006), acrylonitrile (El Hadri, et al. 2005), urethane (Hoffler, et al. 2003, Hoffler and Ghanayem 2005), acetaminophen (Lee, et al. 1996, Zaher, et al. 1998), and ethanol (Bardag-Gorce, et al. 2000).
- Humanized Cyp2E1 mice. Two transgenic mice with human Cyp2E1 have been created. The first mouse reproduces and develops normally, and demonstrates Cyp2E1-dependent toxicity (Morgan, et al. 2002). However, these mice express human and endogenous Cyp2E1, which is not ideal. A true ‘humanized’ Cyp2E1 transgenic mouse was produced by the Gonzalez lab in which the endogenous Cyp2E1 gene was replaced with the human Cyp2E1 gene (Cheung, et al. 2005, Cheung and Gonzalez 2008). Studies in these mice are conducted in order to provide evidence that the Cyp2E1-dependent effects observed in experimental animals will also occur in humans.
- 2-Piperidone. Z-Piperidone is a newly proposed biomarker of Cyp2E1 activity that is detected in urine (Cheng, et al. 2013).
Domain of Applicability
Taxonomic applicability: The Cyp2E1 gene is present across a variety of taxa including humans and primates, mice and rats. AceView (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html) indicates high levels of Cyp2E1 expression from RNA-seq experiments in liver across primate species. Cype2E1 is also present in frogs (Fort, et al. 2003, Saito, et al. 1997) and fish (Howarth, et al. 2011).
Life stages: Studies are primarily on adult liver tissues.
Sex applicability: Cyp2E1 is expressed in both males and females.
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
A variety of substrates have been described (Lieber 1997, Tanaka, et al. 2000). There are >85 known Cyp2E1 substrates. They are low molecular weight compounds, including: molecular oxygen, acetone (Bondoc, et al. 1999), acetaminophen (Lee, et al. 1996, Zaher, et al. 1998), carbon tetrachloride (Wong, et al. 1998), pyrazole, vinyl chloride, furan, chloroform, ethanol (Bardag-Gorce, et al. 2000), benzene (Powley and Carlson 2001), acrylonitrile (El Hadri, et al. 2005), trichloroethylene (Kim and Ghanayem 2006), aniline, N-nitrosodimethylamine, N-nitrosodiethylamine, diethylnitrosamine, thioacetamide (Chilakapati, et al. 2007), urethane (Hoffler, et al. 2003, Hoffler and Ghanayem 2005), and toluene.
Bardag-Gorce, F., Yuan, Q.X., Li, J., French, B.A., Fang, C., Ingelman-Sundberg, M., French, S.W., 2000. The effect of ethanol-induced cytochrome p4502E1 on the inhibition of proteasome activity by alcohol. Biochem. Biophys. Res. Commun. 279, 23-29.
Bondoc, F.Y., Bao, Z., Hu, W.Y., Gonzalez, F.J., Wang, Y., Yang, C.S., Hong, J.Y., 1999. Acetone catabolism by cytochrome P450 2E1: studies with CYP2E1-null mice. Biochem. Pharmacol. 58, 461-463.
Cederbaum, A.I., 2014. Methodology to assay CYP2E1 mixed function oxidase catalytic activity and its induction. Redox Biol. 2C, 1048-1054.
Cheng, J., Chen, C., Kristopher, K.W., Manna, S.K., Scerba, M., Friedman, F.K., Luecke, H., Idle, J.R., Gonzalez, F.J., 2013. Identification of 2-piperidone as a biomarker of CYP2E1 activity through metabolomic phenotyping. Toxicol. Sci. 135, 37-47.
Cheung, C., Gonzalez, F.J., 2008. Humanized mouse lines and their application for prediction of human drug metabolism and toxicological risk assessment. J. Pharmacol. Exp. Ther. 327, 288-299.
Chilakapati, J., Korrapati, M.C., Shankar, K., Hill, R.A., Warbritton, A., Latendresse, J.R., Mehendale, H.M., 2007. Role of CYP2E1 and saturation kinetics in the bioactivation of thioacetamide: Effects of diet restriction and phenobarbital. Toxicol. Appl. Pharmacol. 219, 72-84.
El Hadri, L., Chanas, B., Ghanayem, B.I., 2005. Comparative metabolism of methacrylonitrile and acrylonitrile to cyanide using cytochrome P4502E1 and microsomal epoxide hydrolase-null mice. Toxicol. Appl. Pharmacol. 205, 116-125.
Fort, D.J., McLaughlin, D.W., Rogers, R.L., Buzzard, B.O., 2003. Evaluation of the developmental toxicities of ethanol, acetaldehyde, and thioacetamide using FETAX. Drug Chem. Toxicol. 26, 23-34.
Girre, C., Lucas, D., Hispard, E., Menez, C., Dally, S., Menez, J.F., 1994. Assessment of cytochrome P4502E1 induction in alcoholic patients by chlorzoxazone pharmacokinetics. Biochem. Pharmacol. 47, 1503-1508.
Gonzalez, F.J., 2007. The 2006 Bernard B. Brodie Award Lecture. Cyp2e1. Drug metabolism and disposition: the biological fate of chemicals 35, 1-8.
Hoffler, U., El-Masri, H.A., Ghanayem, B.I., 2003. Cytochrome P450 2E1 (CYP2E1) is the principal enzyme responsible for urethane metabolism: comparative studies using CYP2E1-null and wild-type mice. J. Pharmacol. Exp. Ther. 305, 557-564.
Hoffler, U., Ghanayem, B.I., 2005. Increased bioaccumulation of urethane in CYP2E1-/- versus CYP2E1+/+ mice. Drug Metab. Dispos. 33, 1144-1150.
Howarth, D.L., Passeri, M., Sadler, K.C., 2011. Drinks Like a Fish: Using Zebrafish to Understand Alcoholic Liver Disease. Alcohol. Clin. Exp. Res. 35, 826-829.
Ingelman-Sundberg, M., Johansson, I., Penttila, K.E., Glaumann, H., Lindros, K.O., 1988. Centrilobular expression of ethanol-inducible cytochrome P-450 (IIE1) in rat liver. Biochem. Biophys. Res. Commun. 157, 55-60.
Kim, D., Ghanayem, B.I., 2006. Comparative metabolism and disposition of trichloroethylene in Cyp2e1-/-and wild-type mice. Drug Metab. Dispos. 34, 2020-2027.
Koop, D.R., 1986. Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a. Mol. Pharmacol. 29, 399-404.
Koop, D.R., Laethem, C.L., Tierney, D.J., 1989. The utility of p-nitrophenol hydroxylation in P450IIE1 analysis. Drug Metab. Rev. 20, 541-551.
Lee, S.S., Buters, J.T., Pineau, T., Fernandez-Salguero, P., Gonzalez, F.J., 1996. Role of CYP2E1 in the hepatotoxicity of acetaminophen. J. Biol. Chem. 271, 12063-12067.
Lieber, C.S., 1997. Cytochrome P-4502E1: its physiological and pathological role. Physiol. Rev. 77, 517-544.
Morishima, Y., Peng, H-M., Lin, H-L., Hollenberg, P.F., Sunahara, R., K., Osawa, Y., Pratt, W.B. Regulation of Cytochrome P450 2E1 by Heat Shock Protein 90-Dependent Stabilization and CHIP-Dependent Proteasomal Degradation. 2005. Biochemistry. 44, 49, 16333-16340.
Morgan, K., French, S.W., Morgan, T.R., 2002. Production of a cytochrome P450 2E1 transgenic mouse and initial evaluation of alcoholic liver damage. Hepatology 36, 122-134.
Powley, M.W., Carlson, G.P., 2001. Hepatic and pulmonary microsomal benzene metabolism in CYP2E1 knockout mice. Toxicology 169, 187-194.
Reinke, L.A., Moyer, M.J., 1985. p-Nitrophenol hydroxylation. A microsomal oxidation which is highly inducible by ethanol. Drug Metab. Dispos. 13, 548-552.
Saito, H., Ohi, H., Sugata, E., Murayama, N., Fujita, Y., Higuchi, S., 1997. Purification and characterization of a cytochrome P450 from liver microsomes of Xenopus laevis. Arch. Biochem. Biophys. 345, 56-64.
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.
Tanaka, E., Terada, M., Misawa, S., 2000. Cytochrome P450 2E1: its clinical and toxicological role. J. Clin. Pharm. Ther. 25, 165-175.
Tsutsumi, M., Lasker, J.M., Shimizu, M., Rosman, A.S., Lieber, C.S., 1989. The intralobular distribution of ethanol-inducible P450IIE1 in rat and human liver. Hepatology 10, 437-446.
Wu, D., Cederbaum, A.I., 2005. Oxidative stress mediated toxicity exerted by ethanol-inducible CYP2E1. Toxicol. Appl. Pharmacol. 207, 70-76.
Zaher, H., Buters, J.T., Ward, J.M., Bruno, M.K., Lucas, A.M., Stern, S.T., Cohen, S.D., Gonzalez, F.J., 1998. Protection against acetaminophen toxicity in CYP1A2 and CYP2E1 double-null mice. Toxicol. Appl. Pharmacol. 152, 193-199.