Carcinogenesis, Vol 19, 321-329, Copyright © 1998 by Oxford University Press
HL Lin, ES Roberts and PF Hollenberg
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.
ARTICLES
Heterologous expression of rat P450 2E1 in a mammalian cell line: in situ metabolism and cytotoxicity of N-nitrosodimethylamine
Department of Pharmacology, University of Michigan, Ann Arbor 48109- 0632, USA.
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