Cytochrome P4501A2 constitutively expressed from transduced DNA mediates metabolic activation and DNA-adduct formation of aromatic amine carcinogens in NIH 3T3 cells.
We transduced mouse cytochrome P4501A2 DNA into NIH 3T3 cells by retrovirus-mediated gene transfer. The capacity of the transduced cytochrome P4501A2 for metabolic activation and DNA-carcinogen adduct formation of aromatic amine carcinogens was investigated. Clones of NIH 3T3 cells that constitutively express cytochrome P4501A2 and controls were exposed to a prototype food-derived carcinogenic heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and an aromatic amine, 2-acetylaminofluorene (AAF), and their genomic DNAs were analyzed for adducts by 32P-postlabeling assays. Kinetic analysis of DNA-carcinogen adducts indicated that adduct formation was dependent on the level of the enzyme, the dose of carcinogen, and the duration of exposure. Addition of 7,8-benzoflavone, an inhibitor of P4501A2, blocked both the enzyme activity and DNA-adduct formation, indicating the specific role of P4501A2 in metabolic activation and adduct formation. Three specific IQ-DNA adducts were detected in cells expressing P4501A2. Fingerprints of the in situ DNA adducts were similar to those of the in vivo adducts in rodent hepatic DNA after the administration of IQ. A single AAF-DNA adduct was observed in cells exposed to AAF, but other minor adducts were also detected in vivo. These results show that cells expressing constitutive levels of single cytochrome P450s provide an excellent in situ model system for analyzing the catalytic specificity, metabolic activation, and genotoxicity of putative toxic, mutagenic, and carcinogenic substances.
Battula
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N
Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, Maryland.
Schut
H A
HA
Thorgeirsson
S S
SS
D016475:3T3 Cells; D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D002273:Carcinogens / Q000378:metabolism*; D003001:Cloning, Molecular; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D066298:In Vitro Techniques; D051379:Mice; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011804:Quinolines / Q000378:metabolism*; D051381:Rats; D014162:Transfection
D001571:Benzoflavones; D002273:Carcinogens; D011804:Quinolines; C029216:2-amino-3-methylimidazo(4,5-f)quinoline; C011512:alpha-naphthoflavone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D015660:7-Alkoxycoumarin O-Dealkylase; D019388:Cytochrome P-450 CYP1A2