Inactivation and degradation of human cytochrome P4502E1 by CCl4 in a transfected HepG2 cell line.
Treatment with CCl4 in vivo labilizes cytochrome P4502E1, inactivating the enzyme and enhancing its degradation. To investigate the mechanism of CCl4-induced degradation of human CYP2E1, a recently-established MVh2E1-9 cell line, which constitutively expresses the human CYP2E1 in HepG2 cells was used. CCl4 inhibited oxidation of p-nitrophenol in isolated microsomes from MVh2E1-9 cells suggesting that CCl4 could be metabolized in vitro by the system; however, CCl4 did not promote lipid peroxidation under these conditions. Treatment of the MVh2E1-9 cells in situ with 2 mM CCl4 for 24 hr caused a 30 to 50% loss of both enzyme activity and 2E1 protein. Treatment with cycloheximide at the same time to inhibit constitutive protein synthesis showed a more prominent loss of 2E1 activity and protein. CCl4-induced degradation of CYP2E1 could be prevented by ligands and substrates of 2E1. N-acetylcysteine, N-t-butyl-alpha-phenylnitrone or propylgallate did not significantly prevent CCl4-induced inactivation or degradation of 2E1. After treatment with 14C-labeled CCl4, there was increased radioactive adduct formation in MVh2E1-9 cells compared to control cells lacking CYP2E1. This increase was completely prevented by 4-methylpyrazole and ethanol indicating its dependence on CYP2E1. These results suggest that the human CYP2E1 expressed in the MVh2E1-9 cell line metabolizes CCl4, generating reactive species at the active site that directly inactivate the enzyme and also labilize P450 for degradation by proteases present in the HepG2 cells. Lipid peroxidation is not required for the CCl4-induced inactivation and degradation of CYP2E1 in these cells.
Department of Biochemistry, Mount Sinai School of Medicine, City University of New York, New York, USA.
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Dai
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Cederbaum
D000082:Acetaminophen / Q000494:pharmacology; D002251:Carbon Tetrachloride / Q000494:pharmacology*; D002460:Cell Line; D019392:Cytochrome P-450 CYP2E1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D008861:Microsomes / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D065607:Cytochrome P-450 Enzyme Inhibitors; D009596:Nitrophenols; D000082:Acetaminophen; D003577:Cytochrome P-450 Enzyme System; D002251:Carbon Tetrachloride; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D013755:Tetradecanoylphorbol Acetate; C024836:4-nitrophenol