METABOLISM OF PROMUTAGENS CATALYZED BY DROSOPHILA-MELANOGASTER CYP6A2ENZYME IN SACCHAROMYCES-CEREVISIAE

The somatic mutation and recombination test (SMART) in Drosophila mela nogaster allows screening of chemicals for genotoxicity in a multicell ular organism. In order to correlate data obtained in the SMART with t hose from genotoxicity tests in rodents, it is important to learn more on the variety of drug-metabolizing enzymes present in this insect an d to identify their substrate specificities. In this study we have con centrated on the phase I enzyme cytochrome P450 6A2, which is the firs t cytochrome P450 cloned from Drosophila. A genomic CYP6A2 DNA fragmen t and its corresponding cDNA were cloned and sequenced, revealing a pr eviously unidentified intron with an in frame stop codon. This intron is invariantly present in an insecticide resistant [OR(R)] and a sensi tive (flr(3)) strain. Developmental Northern analysis of CYP6A2 mRNA d emonstrated a peak of expression in the third larval and pupal stage. CYP6A2 mRNA was found to be present in the insecticide-resistant strai n at higher levels than in the insecticide-sensitive strain. Therefore , insecticide resistance might be correlated with enhanced CYP6A2 expr ession. The substrate specificity of CYP6A2 enzyme was investigated by coexpressing CYP6A2 cDNA with the cDNA for human NADPH-cytochrome P45 0 reductase in the yeast Saccharomyces cerevisiae. The transformed str ain activated the mycotoxin aflatoxin B-1 to a product that induced ge ne conversion, scored at the trp5 locus. Two other compounds, 7,12-dim ethylbenz[a]anthracene (DMBA) and 3-amino-1-methyl-5H-pyrido[4,3-b]ind ole (Trp-P-2), were metabolized in the transformed strain to cytotoxic products. (C) 1996 Wiley-Liss, Inc.