Metabolism of promutagens catalyzed by Drosophila melanogaster CYP6A2 enzyme in Saccharomyces cerevisiae.
The somatic mutation and recombination test (SMART) in Drosophila melanogaster allows screening of chemicals for genotoxicity in a multicellular organism. In order to correlate data obtained in the SMART with those from genotoxicity tests in rodents, it is important to learn more on the variety of drug-metabolizing enzymes present in this insect and to identify their substrate specificities. In this study we have concentrated on the phase I enzyme cytochrome P450 6A2, which is the first cytochrome P450 cloned from Drosophila. A genomic CYP6A2 DNA fragment and its corresponding cDNA were cloned and sequenced, revealing a previously unidentified intron with an inframe stop codon. This intron is invariantly present in an insecticide resistant [OR(R)] and a sensitive (flr3) strain. Developmental Northern analysis of CYP6A2 mRNA demonstrated a peak of expression in the third larval and pupal stage. CYP6A2 mRNA was found to be present in the insecticide-resistant strain at higher levels than in the insecticide-sensitive strain. Therefore, insecticide resistance might be correlated with enhanced CYP6A2 expression. The substrate specificity of CYP6A2 enzyme was investigated by coexpressing CYP6A2 cDNA with the cDNA for human NADPH-cytochrome P450 reductase in the yeast Saccharomyces cerevisiae. The transformed strain activated the mycotoxin aflatoxin B1 to a product that induced gene conversion, scored at the trp5 locus. Two other compounds, 7,12-dimethylbenz[a]anthracene (DMBA) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), were metabolized in the transformed strain to cytotoxic products.
Saner
C
C
Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach, Switzerland.
Weibel
B
B
Wurgler
F E
FE
Sengstag
C
C
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000493:pharmacokinetics; D016604:Aflatoxin B1 / Q000493:pharmacokinetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001711:Biotransformation / Q000235:genetics*; D002243:Carbolines / Q000493:pharmacokinetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D005785:Gene Conversion / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental; D017344:Genes, Insect; D005813:Genes, Synthetic; D006801:Humans; D007438:Introns; D007814:Larva; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009153:Mutagens / Q000493:pharmacokinetics*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D009928:Organ Specificity; D011355:Prodrugs / Q000493:pharmacokinetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D002243:Carbolines; D009153:Mutagens; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D015127:9,10-Dimethyl-1,2-benzanthracene; C019133:3-amino-1-methyl-5H-pyrido(4,3-b)indole; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D009251:NADPH-Ferrihemoprotein Reductase