High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase

Abstract

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-0-deethylase activity, a problem which is related to this specific substrate. Using the microsomes, it was demonstrated that aflatoxin B, 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-l-methyl-5//-pyrido[4,3-ft]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-dimethyUmidazo[4,5-/]quinoUne (MelQ) and 2-amino-3,8-dimethyUmidazo[4,5-/]quinoxaline (MelQ*). MeIQ x was activated efficiently by both CYP enzymes, whereas MelQ 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.

Recombinant human P450 forms 1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens in Escherichia coli lacZ strains

“…This result is consistent with most of the earlier data, although previously applied assays may have had insufficient sensitivity to detect bioactivation by 1A1 or 1B1. The exceptional result of Sengstag et al [1994], who found that P450 forms 1A1 and 1A2 were similarly effective for activation of MeIQx, is inconsistent with our findings.…”

DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes

“…Antigenic proteins have been detected in the transformed strains, and enzyme activity specific for the produced enzymes has been measured [27-301. In addition, microsomes prepared from the strains efficiently activated various promutagens to potentSalmonella mutagens [31]. Here we demonstrate that some of the drugs are also activated in intact yeast cells to products that significantly induce recombination.…”

Expression of a Human Cytochrome P450 in Yeast Permits Analysis of Pathways for Response to and Repair of Aflatoxin-Induced DNA Damage

“…S9 fractions of yeast strains expressing hCYP1A2 or empty vector were prepared as previously described (86), except that growth conditions were modified to match those used in the cytotoxicity and mutagenicity studies. Specifically, 10 6 cells were grown for 7 h in 250 ml of synthetic media lacking uracil.…”