Summary
CYP2E1, a member of the cytochrome P450 family, is induced by ethanol. CYP2E1 activates many hepatotoxins to their reactive toxic intermediate form, and generates reactive oxygen species (ROS) during its catalytic cycle. Induction of CYP2E1 plays an important role in ethanol-induced oxidant stress and ethanol toxicity. To study the biochemical and toxicological properties of CYP2E1, our laboratory developed a HepG2 cell line which constitutively expresses the human CYP2E1 form. These cells displayed elevated oxidative stress, loss of mitochondrial function and loss of viability when challenged with prooxidants such as ethanol, polyunsaturated fatty acids (PUFA) such as arachidonic acid, iron, or when depleted of the critical antioxidant glutathione, as compared with control HepG2 cells which do not express CYP2E1. In the sections below , protocols are described for use of these cell lines to assay for CYP2E1-dependent oxidant stress and toxicity. Methods are described as to how the cell lines were established and maintained, how CYP2E1 is assayed, how cellular viability, mitochondrial function and generation of oxidant stress are determined.
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References
1. Guengerich, F. P., Kim, D. H., and Iwasaki, M. (1990) Role of human cytochrome P450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol. 14, 168–179.
2. Yang, C. S., Yoo, J. S.H., Ishizaki, H., and Hong, J. (1990) Cytochrome P450 IIE1: roles in nitrosamine metabolism and mechanism of regulation. Drug Metab. Rev. 22, 147–159.
3. Koop, D. R. (1992) Oxidative and reductive metabolism by cytochrome P4502E1. FASEB J. 6, 724–730.
4. Esktrom, G., and Ingelman-Sundberg, M. (1989) Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P450. Biochem. Pharmacol. 38, 1313–1319.
5. Tsukamoto, H., and Lu, S. C. (2001) Current concepts in the pathogenesis of alcoholic liver injury. FASEB J. 15, 1335–1349.
6. Cederbaum, A. I. (2001) Introduction: serial review: alcohol, oxidative stress and cell injury. Free Rad. Biol. Med. 31, 1524–1526.
7. Castillo, T., Koop, D. R., Kamimura, S., Triadafilopoulos, G., and Tsukamoto, H. (1992) Role of cytochrome P4502E1 in ethanol-, carbon tetrachloride-, and iron-dependent microsomal lipid peroxidation. Hepatology 16, 992–996.
8. Morimoto, M., Zern, M. A., Hagbjork, A. L., Ingelman-Sundberg, M., and French, S. W. (1994) Fish oil, alcohol, and liver pathology: role of cytochrome P4502E1. Proc. Soc. Exp. Biol. Med. 207, 197–205.
9. Nanji, A. A., Zhao, S., Sadrzadeh, S. M.H., Dannenberg, A. J., Tahan, S. T., and Waxman, D. J. (1994) Markedly enhanced cytochrome P4502E1 induction and lipid peroxidation is associated with severe liver injury in fish oil-ethanol-fed rats. Alcohol. Clin. Exp. Res. 18, 1280–1285.
10. Gouillon, Z., Lucas, D., Li, J., Hagbjork, A. L., French, B. A., Fu, P., Fang, C., Ingelman-Sundberg, M., Donohue, T. M. Jr., and French, S. W. (2000) Inhibition of ethanol-induced liver disease in the intragastric feeding rat model by chlormethiazole. Proc. Soc. Exp. Biol. Med. 224; 302–308.
11. Koop, D. R., Klopfenstein, B., Iimuro, Y., and Thurman, R. G. (1997) Gadolinium chloride blocks alcohol-dependent liver toxicity in rats treated chronically with intragastric alcohol despite the induction of CYP2E1. Mol. Pharmacol. 51, 944–950.
12. Kono, H., Bradford, B. U., Yin, M., Sulik, K. K., Koop, D. R., Peters, J. M., Gonzalez, F. J., McDonald, T., Dikalova, A., Kadiiska, M. B., et al. (1999) CYP2E1 is not involved in early alcohol-induced liver injury. Am. J. Physiol. 277, G1259-G1267.
13. Isayama, F., Froh, M., Bradford, B. U. Mckim, S. E., Kadiiska, M. B., Connor, H. D., Mason, R. P., Koop, D. R., Wheeler, M. D., and Arteel, G. E. (2003) The CYP inhibitor 1-aminobenzotriazole does not prevent oxidative stress associated with alcohol-induced liver injury in rats and mice. Free Rad. Biol. Med. 35, 1568–1581.
14. Morgan, K., French, S. W., and Morgan, T. R. (2002) Production of a cytochrome P450 2E1 transgenic mouse and initial evaluation of alcoholic liver damage. Hepatology. 36, 122–134.
15. Bardag-Gorce, F., Yuan, Q. X., Li, J., French, B. A., Fang, C., Ingelman-Sundberg, M., and French, S. W. (2000) The effect of ethanol-induced cytochrome P450 2E1 on the inhibition of proteasome activity by alcohol. Biochem. Biophys. Res. Commun. 279, 23–29.
16. Bradford, B. U., Kono, H., Isayama, F., Kosyk, O. Wheeler, M. D., Akiyama, T. E., et al (2005) Cytochrome P4502E1 but not NADPH oxidase is required for ethanol-induced oxidative DNA damage in rodent liver. Hepatology 41, 336–344.
17. Tanaka, E., Terada, M., and Misawa, S. (2000) Cytochrome P450 2E1: its clinical and toxicological role. J. Clin. Pharm. Ther. 25, 165–175.
18. Gonzalez, F. J. (2005) Role of cytochromes P450 in chemical toxicology and oxidative stress I studies with CYP2E1. Mutat. Res. 569, 101–110.
19. Dai, Y., Rashba-Step, J., and Cederbaum, A. I. (1993) Stable transfection of human cytochrome P4502E1 in HepG2 cells: characterization of catalytic activities and production of reactive oxygen intermediates. Biochemistry 32, 6928–6937.
20. Chen, Q., and Cederbaum, A. I. (1998) Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in HepG2 cells. Mol. Pharmacol. 53, 638–648.
21. Dai, Y., and Cederbaum, A. I. (1995) Cytotoxicity of acetaminophen in human cytochrome P4502E1-transfected HepG2 cells. J. Pharmacol. Exp. Ther. 273, 1497–1505.
22. Dai, Y., and Cederbaum, A. I. (1995) Inactivation and degradation of human cytochrome P4502E1 by CCl4 in a transfected HepG2 cell line. J. Pharmacol. Exp. Ther. 275, 1614–1622.
23. Wu, D., and Cederbaum, A. I. (1996) Ethanol toxicity to transfected HepG2 cells expressing human cytochrome P4502E1. J. Biol. Chem. 271, 23914–23919.
24. Chen, Q., Galleano, M., and Cederbaum, A. I. (1997) Cytotoxicity and apoptosis produced by arachidonic acid in HepG2 cells overexpressing human cytochrome P4502E1. J. Biol. Chem. 272, 14532–14541.
25. Wu, D., and Cederbaum, A. I. (1999) Ethanol-induced apoptosis to hepatic cell lines expressing human cytochrome P4502E1. Alcohol. Clin. Exp. Res. 23, 67–76.
26. Sakurai, K., and Cederbaum, A. I. (1998) Oxidative stress and cytotoxicity induced by ferric-nitrilotriacetate in HepG2 cells expressing CYP2E1. Mol. Pharmacol. 54, 1024–1035.
27. Wu, D., and Cederbaum, A. I. (2001) Removal of glutathione produces apoptosis and necrosis in HepG2 cells overexpressing CYP2E1. Alcohol. Clin. Exp. Res. 25, 619–628.
28. Caro, A. A., and Cederbaum, A. I. (2004) Oxidative stress, toxicology and Pharmacology of CYP2E1. Annu. Rev. Pharmacol. Toxicol. 44, 27–42.
29. Kessova, I. and Cederbaum, A. I. (2003) CYP2E1: Biochemistry, toxicology and regulation. Curr. Mol. Med. 3, 509–518.
30. Jimenez-Lopez, J. M., and Cederbaum, A. I. (2005) CYP2E1-dependent oxidative stress and toxicity: role in ethanol-induced liver injury. Expert Opinion Drug Metab. Toxicol. 1, 671–685.
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Wu, D., Cederbaum, A.I. (2008). Development and Properties of HepG2 Cells That Constitutively Express CYP2E1. In: Nagy, L.E. (eds) Alcohol. Methods in Molecular Biology™, vol 447. Humana Press. https://doi.org/10.1007/978-1-59745-242-7_11
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DOI: https://doi.org/10.1007/978-1-59745-242-7_11
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