High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase.
Yeast Saccharomyces cerevisiae strains have been constructed that co-express cDNAs coding for the human cytochrome P-450 enzymes CYP1A1 or CYP1A2 in combination with human NADPH-cytochrome P-450 reductase (oxidoreductase). Microsomal fractions prepared from the strains were able to efficiently activate various drugs to Salmonella mutagens. These experiments demonstrated that a functional interaction occurred between the respective human enzymes in the yeast microsomes. For every drug tested, the microsomes containing CYP enzymes and oxidoreductase were 2- to 4-fold better in activation than the corresponding microsomes that contained CYP alone. Interestingly, co-expression of CYP1A2 with oxidoreductase resulted in a decrease of 7-ethoxyresorufin-O-deethylase activity, a problem which is related to this specific substrate. Using the microsomes, it was demonstrated that aflatoxin B1 was activated to a mutagen not only by CYP1A2 but also by CYP1A1. In contrast, benzo[a]pyrene was exclusively activated by CYP1A1 whereas CYP1A2 was inactive. The drug 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was activated by CYP1A2 and to a lesser extent by CYP1A1. A strong substrate specificity was observed with the two structurally related heterocyclic arylamines 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). MeIQx was activated efficiently by both CYP enzymes, whereas MeIQ was only activated by CYP1A2 and not by CYP1A1. The fact that microsomes from vector transformed control strains were unable to activate any of the drugs studied underlines the suitability of these microsomes for metabolic studies. Moreover, the presence of suitable marker genes in the yeast strains will enable us to study mitotic recombination and gene conversion events induced by drugs that require metabolic activation.
Sengstag
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Institute of Toxicology, Swiss Federal Institute of Technology and University of Zürich.
Eugster
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Würgler
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D001483:Base Sequence; D001711:Biotransformation; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008861:Microsomes / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D009153:Mutagens / Q000493:pharmacokinetics*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011355:Prodrugs / Q000493:pharmacokinetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure*; D014170:Transformation, Genetic
D018076:DNA, Complementary; D009153:Mutagens; D011355:Prodrugs; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D009251:NADPH-Ferrihemoprotein Reductase