Heterologous expression of rat P450 2E1 in a mammalian cell line: in situ metabolism and cytotoxicity of N-nitrosodimethylamine.
GM0637, a human fibroblast cell line, was transfected with pCMV2E1, an expression vector containing the full length cDNA for rat cytochrome P450 2E1 (P450 2E1), and with pCMVneo, which contained vector alone, and the selected clones were designated GM2E1 and GMneo, respectively. Western blot analysis showed that GM2E1, but not GMneo, expressed a protein that reacted with anti-human P450 2E1 antibody. The 7-ethoxycoumarin O-deethylase,p-nitrophenol hydroxylase, and N-nitrosodimethylamine (NDMA) demethylase activities of the P450 in these cells were measured in monolayer cell cultures without preparing microsomes. Exposure of the GM2E1 cells to NDMA for 4 days caused severe decreases in cell viability, as determined by crystal violet uptake, and showed a sigmoidal dose-response curve with a median lethal dose of 17 microM. In contrast, the viability of GMneo cells was not altered by NDMA even at concentrations up to 10 mM. Time- and concentration-dependent methylation of DNA, RNA and protein by [14C]NDMA was only observed in cells expressing P450 2E1. Inhibitors of P450 2E1 activity such as ethanol, 4-methylpyrazole, and isoniazid caused a 90% decrease in the methylation of cellular macromolecules and also completely protected the cells against NDMA-mediated toxicity. The cytotoxicity due to exposure to NDMA was partially inhibited by antioxidants such as N-acetylcysteine, ascorbic acid, butylated hydroxyanisole and N-t-butyl-alpha-phenylnitrone but was not potentiated upon glutathione depletion. These results document the ability of rat P450 2E1 to metabolize NDMA to toxic reactive intermediates and demonstrate that this cell line provides a useful model for studying the mechanisms of metabolism-mediated toxicity and carcinogenesis.
Lin
H L
HL
Department of Pharmacology, University of Michigan, Ann Arbor 48109-0632, USA.
Roberts
E S
ES
Hollenberg
P F
PF
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D015153:Blotting, Western; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003374:Coumarins / Q000493:pharmacokinetics; D003497:Cyclic N-Oxides; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D000077604:Fomepizole; D005982:Glutathione Transferase / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007538:Isoniazid / Q000494:pharmacology; D009589:Nitrogen Oxides / Q000494:pharmacology; D009596:Nitrophenols / Q000493:pharmacokinetics; D009603:Nitroso Compounds / Q000493:pharmacokinetics* / Q000633:toxicity*; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D013113:Spin Labels; D013997:Time Factors; D014162:Transfection
D000975:Antioxidants; D003374:Coumarins; D003497:Cyclic N-Oxides; D004137:Dinitrochlorobenzene; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009589:Nitrogen Oxides; D009596:Nitrophenols; D009603:Nitroso Compounds; D011720:Pyrazoles; D013113:Spin Labels; C008651:4-nitrosodimethylaniline; C017299:7-ethoxycoumarin; C029217:phenyl-N-tert-butylnitrone; D000431:Ethanol; D019328:Buthionine Sulfoximine; D000077604:Fomepizole; D019392:Cytochrome P-450 CYP2E1; D005982:Glutathione Transferase; D007538:Isoniazid; C024836:4-nitrophenol