Stable expression of human CYP2E1 in Chinese hamster cells: high sensitivity to N,N-dimethylnitrosamine in cytotoxicity testing.
Involvement of human CYP2E1 expressed in genetically engineered cells in the metabolic activation of promutagens and procarcinogens was studied. An expression plasmid containing an insert of CYP2E1 cDNA and SR alpha promoter was constructed and transfected into the cultured cell line CR-119 which had previously been established by introducing a cDNA coding for NADPH-cytochrome P450 reductase. Among newly established cell lines, ER-181 showed the highest expression of CYP2E1 mRNA. Production of the CYP2E1 protein was confirmed by Western blot analysis using anti-rat CYP2E1 antibodies. Assay of 7-ethoxycoumarin O-deethylase activity demonstrated that ER-181 cells acquired the catalytic function of CYP2E1. ER-181 cells showed higher sensitivity to N,N-dimethylnitorosamine (DMN) in cytotoxicity assays as compared to parental CR-119 cells. Hypersensitivity to DMN of ER-181 cells was completely suppressed by 3-amino-1,2,4-triazole, a known inhibitor of CYP2E1. These results indicate that ER-181 cells which express human CYP2E1 are a useful tool to investigate toxicological functions of the cytochrome.
Nakagawa
T
T
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University N12, Sapporo, Japan.
Sawada
M
M
Gonzalez
F J
FJ
Yokoi
T
T
Kamataki
T
T
D000818:Animals; D015153:Blotting, Western; D002273:Carcinogens / Q000633:toxicity; D002460:Cell Line; D006224:Cricetinae; D003470:Culture Media; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004128:Dimethylnitrosamine / Q000633:toxicity*; D006801:Humans; D009152:Mutagenicity Tests / Q000379:methods; D009153:Mutagens / Q000633:toxicity; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014468:Umbelliferones / Q000378:metabolism
D002273:Carcinogens; D003470:Culture Media; D009153:Mutagens; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014468:Umbelliferones; C031477:7-hydroxycoumarin; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D004128:Dimethylnitrosamine