Ethanol cytotoxicity to a transfected HepG2 cell line expressing human cytochrome P4502E1.
The effect of ethanol on the viability of a HepG2 cell model which was developed to constitutively express human CYP2E1 was studied in an attempt to establish a linkage between CYP2E1, reactive oxygen intermediates, and ethanol toxicity. Assays of toxicity included leakage of lactate dehydrogenase, trypan blue uptake, morphology, and formazan production. Ethanol was toxic to HepG2 E9 cells, which express CYP2E1, but not to HepG2 MV5 cells, which do not express CYP2E1. The ethanol toxicity was dependent on the concentration of ethanol, starting with 10 m ethanol, and on the time of incubation with ethanol. Phorbol 12-myristate 13-acetate, which increases the expression of CYP2E1 in this model, increased the toxicity by ethanol. Ethanol toxicity was prevented by 4-methylpyrazole and by diallyl sulfide, inhibitors of CYP2E1. The ethanol toxicity was also prevented by radical trapping agents such as N-acetylcysteine and N-t-butyl-alpha-phenylnitrone, antioxidative agents such as catalase, superoxide dismutase, thiourea, and uric acid, and inhibitors of lipid peroxidation, such as vitamin E phosphate, Trolox, and diphenylphenylenediamine. Besides ethanol, other substrates such as Me2SO, CCl4, isoniazid, and N,N-dimethylnitrosamine were cytotoxic to cells expressing CYP2E1 but not to control cells. These results indicate that ethanol was toxic to HepG2 cells which express human CYP2E1 by a pathway sensitive to inhibitors of CYP2E1 and to a variety of antioxidative agents. This model appears to be useful in efforts to establish a CYP2E1-dependent ethanol hepatotoxicity system and to evaluate the role of oxidative stress and reactive radical species in the toxicity by ethanol.
Wu
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Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Cederbaum
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AI
D000498:Allyl Compounds; D002460:Cell Line; D019392:Cytochrome P-450 CYP2E1 / Q000502:physiology*; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004790:Enzyme Induction; D000431:Ethanol / Q000633:toxicity*; D000077604:Fomepizole; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D011720:Pyrazoles / Q000494:pharmacology; D011994:Recombinant Proteins; D013440:Sulfides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013890:Thiourea / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014527:Uric Acid / Q000494:pharmacology
D000498:Allyl Compounds; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D011720:Pyrazoles; D011994:Recombinant Proteins; D013440:Sulfides; D014527:Uric Acid; D000431:Ethanol; C038491:allyl sulfide; D000077604:Fomepizole; D019392:Cytochrome P-450 CYP2E1; D013890:Thiourea; D013755:Tetradecanoylphorbol Acetate