Stable expression of human cytochrome P450IIE1 in mammalian cells: metabolic activation of nitrosodimethylamine and formation of adducts with cellular DNA.
To introduce cytochrome P450IIE1 DNA stably into the chromosomal DNA of mammalian cells, we constructed recombinant retroviruses containing the full-length complementary DNA for human cytochrome P450IIE1 and a selectable neo gene. Rat and mouse cells were infected with these viruses, and clones expressing the neo marker gene product were selected in G418-containing medium. Analysis of the DNA of the clones by Southern blotting showed that the viral DNA was integrated into the cellular DNA. Enzymatic analysis of the clones showed that the transduced DNA directed the expression of enzymatically active cytochrome P450IIE1. Treatment of the cells with the carcinogen [14C]-nitrosodimethylamine and analysis of the cellular DNA by CsCl equilibrium density gradients showed incorporation of the label into DNA, indicating the formation of covalent adducts with the cell DNA. Construction of recombinant cell lines constitutively expressing cytochrome P450IIE1 provides a permanent source for this enzyme and can aid in the analysis of its catalytic properties, such as the metabolic activation of chemical mutagens/carcinogens, and the consequent cytotoxic and genotoxic effects of these compounds.
Nouso
K
K
Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Thorgeirsson
S S
SS
Battula
N
N
D016475:3T3 Cells; D000818:Animals; D001711:Biotransformation; D002478:Cells, Cultured; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004128:Dimethylnitrosamine / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D051379:Mice; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D004128:Dimethylnitrosamine