Liver carcinogen aflatoxin B1 as an inducer of mitotic recombination in a human cell line.

The mycotoxin aflatoxin B1 (AFB1) is one of the most potent rodent and human liver carcinogens. Upon cytochrome P450-specific metabolism, it induces mutations as well as mitotic recombination events in in vitro systems. We have found that in the lower eukaryote yeast, the recombinagenic activity of AFB1 surpasses its mutagenic activity, and we speculated on possible consequences in terms of the mechanism of liver carcinogenesis. In this study we investigated whether the recombinagenic activity of AFB1 also would be identified in human cells. To address this question, we followed the fate of a heterozygous thymidine kinase (tk) allele in the human lymphoblastoid cell line TK6 upon exposure to AFB1. Individual mutants that had lost tk activity were subjected to loss of heterozygosity analysis of the tk locus and its flanking markers. Fluorescence in situ hybridization analysis on chromosome 17 also was performed. In parallel, a similar analysis was performed on TK6 cells exposed to the alkylating agent N-nitrosomethylurea, a well-known classic point mutagen. Our analysis showed a difference in the molecular mechanism leading to inactivation of the tk allele upon exposure to these two mutagens. In AFB1-exposed cells the fraction of recombination-derived mutants predominated, whereas in N-nitrosomethylurea-exposed cells the fraction of point mutants was higher. Thus, the recombinagenic activity of AFB1 previously identified in a lower eukaryote also was found in the human cell line TK6. Our data support the hypothesis that mitotic recombination represents a central mechanism of action in AFB1-induced liver carcinogenesis.

The potential use of mutation spectra in cancer related genes in genetic toxicology: a statement of a GUM working group.

In recent years, there has been widespread interest in the relationship between carcinogenic exposure and mutation spectra in cancer-related genes. To evaluate potential benefits and/or limitations in the use of mutation spectra in genetic toxicology, a GUM working group has been established to discuss this subject. Based on methodological possibilities and limitations, the impact of mutation spectra in the interpretation of animal experiments and in the identification of etiological agents in human cancer has been considered. With respect to experimental animals, the analyses of mutation spectra within long-term rodent carcinogenicity studies may provide some additional information on the mode of action of the respective carcinogen, however, the interpretation of results should be done carefully and only in context with other toxicological data available. Regarding human exposure, the analysis of mutation spectra in p53 or ras genes supplies information on the genotoxic properties of the respective agent. Nevertheless, on the individual level, the presence or absence of defined mutations in cancer-related genes in human tumors does not permit a definite conclusion about the causative agent.

Codon 249 of the human TP53 tumor suppressor gene is no hot spot for aflatoxin B1 in a heterologous background.

Mutations in the TP53 tumor suppressor gene are the most common alteration in cancer, and human primary liver cancers related to previous dietary exposure to the mycotoxin aflatoxin B1 (AFB1) exhibit a specific hot spot mutation at TP53 codon 249. We have asked whether the 249 hot spot is related to a particular susceptibility to AFB1 of this TP53 region or whether it is related to a phenotype of the 249S p53 mutant protein. This was addressed by constructing a metabolically competent variant of Saccharomyces cerevisiae strain yIG397 expressing human cytochrome P450 1A2 and P450-reductase and isolating AFB1-induced mutants that failed to express the genomic ADE2 reporter gene. Molecular analysis revealed that only 8/40 mutants had a mutation in the TP53 target gene, whereas 32/40 mutants were due to a recombination event eliminating the ADE2 reporter gene. None of 19 mutations identified in the eight mutant TP53 plasmids altered codon 249, thus this codon was no hot spot if the TP53 gene was in the heterologous background yeast. The genotoxic action of AFB1 was completely different from that of the alkylating agent ethyl-methane-sulfonate, where 28/30 induced mutations were linked to the TP53 target gene.

Heterocyclic aromatic amines efficiently induce mitotic recombination in metabolically competent Saccharomyces cerevisiae strains.

Heterocyclic aromatic amines (HAs) represent a class of potent bacterial mutagens and rodent carcinogens which gain their biological activity upon metabolic conversion by phase I and phase II enzymes. Subsequent to cytochrome P450 (CYP)-dependent hydroxylation, mainly catalyzed by CYP1A2, acetylation mediated by the activity of N-acetyltransferase, NAT2, produces the ultimate electrophilic product that may react with DNA. In addition to point mutations observed in HA-exposed cells as genotoxic endpoint in vitro, loss of heterozygosity (LOH) has often been identified in HA-related rodent tumors as another endpoint in vivo. LOH may reflect a chromosomal deletion, a chromosome loss or a previous mitotic recombination event and it represents a prominent mechanism for the inactivation of tumor suppressor alleles. In this study we have investigated whether LOH observed in several HA-induced rodent tumors is related to a recombinogenic activity of HA compounds, and to address this question we have studied the genotoxic activity of several HAs in metabolically competent Saccharomyces cerevisiae strains. For this purpose expression vectors have been constructed providing simultaneous expression of three human enzymes, CYP1A2, NADPH-cytochrome P450 oxidoreductase and NAT2 in different genotoxicity tester strains. Evidence for functional expression of all three enzymes has been obtained. One strain allowed us to monitor HA-induced gene conversion, another one HA-induced chromosomal translocation. A third strain allowed us to study HA-induced forward mutations in the endogenous URA3 gene. It was found that 2-amino-3-methylimidazo-[4,5-f]quinoline and 2-amino-3, 8-dimethylimidazo-[4,5-f]quinoxaline produced a strong recombinogenic response in either recombination tester strain. The recombinogenic activity was comparable with the mutagenic activity of the compounds. The other HAs, 2-amino-3, 4-dimethyl-imidazo-[4, 5-f]quinoline, 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole, 2-aminodipyrido-[1,2-a:3', 2'-d]imidazole, 3-amino-1-methyl-5H pyrido-[4,3-b]indole and 2-amino-1-methyl-6-phenyl-imidazo-[4, 5-b]pyridine, produced weak or no increases in the genotoxic endpoints of interest. The described strains may provide a suitable tool to characterize the genotoxic potential of HAs in more detail.

Rearrangements in minisatellite sequences induced by aflatoxin B1 in a metabolically competent strain of Saccharomyces cerevisiae.

The role of aflatoxin B1 (AFB1) in the induction of rearrangements affecting minisatellite sequences was studied in an in vitro yeast model. The Saccharomyces cerevisiae strain used expresses human cytochrome P450 1A2 and NADPH-cytochrome P450 oxidoreductase and has previously been used to study genetic recombination events induced by AFB1. DNA multilocus fingerprinting was performed using probe M13 core hybridizing to a set of hypervariable minisatellite sequences in S. cerevisiae. Frequent spontaneous genomic alterations that affect the minisatellite fingerprint pattern were observed. Control cultures showed 15.8% rearrangements in minisatellites, and this frequency increased to 40.0% in cultures exposed to AFB1 (80 microg/ml). A total of approximately 29 minisatellite loci were visualized for each culture. Given the number of cultures examined (40 AFB1-treated and 38 controls) the rearrangement frequency per detectable minisatellite was 2.59% in the AFB1-treated group and 0.73% in the control group, which represents a statistically significant (P = 0.001) difference. Thus, our data strongly suggest that AFB1 can promote the genetic events responsible for minisatellite rearrangements in the yeast genome. Such genetic rearrangements may be important events during the etiology of liver carcinogenesis in people chronically exposed to dietary aflatoxins.

The Sge1 protein of Saccharomyces cerevisiae is a membrane-associated multidrug transporter.

In this study, we report the further characterization of the Saccharomyces cerevisiae crystal violet-resistance protein Sge1. Sge1 is a highly hydrophobic 59 kDa protein with 14 predicted membrane-spanning domains. It shares homologies with several drug-resistance proteins and sugar transporters of the major facilitator superfamily. Here, we have demonstrated that Sge1 is not only a crystal violet-resistance protein, but that it also confers resistance to ethidium bromide and methylmethane sulfonate. Disruption of SGE1 leads to increased sensitivity towards all three compounds, thus designating Sge1 as a multiple drug-resistance protein. Subcellular fractionation as well as immunolocalization on whole yeast cells demonstrated that Sge1 was tightly associated with the yeast plasma membrane. Furthermore, Sge1 was highly enriched in preparations of yeast plasma membranes. In analogy to other multidrug-resistance proteins, we suggest that Sge1 functions as a drug export permease.

Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.

Genotoxicity of ethyl carbamate (urethane) in Salmonella, yeast and human lymphoblastoid cells.

Ethyl carbamate is a known carcinogen occurring in fermented food and beverages and is therefore of interest for food safety assurance. We studied the genotoxicity of ethyl carbamate in Salmonella typhimurium, in Saccharomyces cerevisiae and in human lymphoblastoid TK6 cells. In absence of cytochrome P450 enzymes, no ethyl carbamate-mediated genotoxicity was observed in any of the three test systems in the non-cytotoxic range. In the presence of an activating system, ethyl carbamate was found to be mutagenic in Salmonella typhimurium strain TA100 but not in strains TA98 and TA102, indicating base-pair substitutions at G-C base pairs. In contrast, no significant mutagenicity of ethyl carbamate could be detected in human lymphoblastoid TK6 cells. However, applied in cytotoxic concentrations, ethyl carbamate was genotoxic for Saccharomyces cerevisiae in the absence of P450-mediated metabolic activation. Inhibitors of P450IIE1 (DMSO, ethanol and dithiodiethylcarbamate) diminished ethyl carbamate-mediated mutagenicity in Salmonella typhimurium strain TA100 in a dose dependent manner, suggesting that P450IIE1 is the activating enzyme.

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells.

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5' and 3' deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the beta-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9 x 10(-4), whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8 x 10(-4). The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.

Genotoxicity of aflatoxin B1: evidence for a recombination-mediated mechanism in Saccharomyces cerevisiae.

The potent liver carcinogen aflatoxin B1 (AFB1) is metabolized by cytochrome P450 to the mutagenic epoxide. We have observed that activated AFB1 also strongly induced mitotic recombination in the yeast Saccharomyces cerevisiae. To compare the recombinogenicity of AFB1 to its mutagenicity, three metabolically competent S. cerevisiae strains have been constructed. The frequencies of induced recombinants resulting from gene conversion or chromosomal translocations were determined by different prototrophic selections using two strains, whereas the inducibility of forward mutations was determined by the frequency of drug resistance in the third strain. Human cytochrome P4501A1- (CYP1A) and NADPH-cytochrome P450-oxidoreductase cDNAs were expressed in the strains to ensure intracellular metabolism to the epoxide. Exposure of the strains to AFB1 resulted in a 139- and 24-fold increase in the translocation and gene conversion frequencies, respectively, whereas the mutation frequency was increased only 3-fold. In contrast, benzo[a]pyrene-7,8-dihydrodiol and ethyl methanesulfonate induced mutation and mitotic recombination to similar degrees. We conclude that AFB1 exerted a strong recombinogenic, but only a weak mutagenic, effect. The recombinogenicity of AFB1 in yeast may indicate a mechanism for the high proportion of loss of heterozygosity that has been detected in AFB1-related human liver cancers.

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.

Identification of the sequences in HMG-CoA reductase required for karmellae assembly.

In all eukaryotic cells that have been examined, specific membrane arrays are induced in response to increased levels of the ER membrane protein, HMG-CoA reductase. Analysis of these inducible membranes has the potential to reveal basic insights into general membrane assembly. Yeast express two HMG-CoA reductase isozymes, and each isozyme induces a morphologically distinct proliferation of the endoplasmic reticulum. The isozyme encoded by HMG1 induces karmellae, which are long stacks of membranes that partially enclose the nucleus. In contrast, the isozyme encoded by HMG2 induces short stacks of membrane that may be associated with the nucleus, but are frequently present at the cell periphery. To understand the molecular nature of the different cellular responses to Hmg1p and Hmg2p, we mapped the region of Hmg1p that is needed for karmellae assembly. For this analysis, a series of exchange alleles was examined in which a portion of the Hmg2p membrane domain was replaced with the corresponding Hmg1p sequences. Results of this analysis indicated that the ER lumenal loop between predicted transmembrane domains 6 and 7 was both necessary and sufficient for karmellae assembly, when present in the context of an HMG-CoA reductase membrane domain. Immunoblotting experiments ruled out the simple possibility that differences in the amounts of the various chimeric HMG-CoA reductase proteins was responsible for the altered cellular responses. Our results are consistent with the hypothesis that each yeast isozyme induces or organizes a qualitatively different organization of ER membrane.

Targeting of heterologous membrane proteins into proliferated internal membranes in Saccharomyces cerevisiae.

Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has previously been observed to induce the proliferation of internal endoplasmic reticulum-like membranes. In order to exploit this amplified membrane surface area for the accommodation of heterologous microsomal proteins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) to sequences encoding the HMG2/1 peptide and expressed the hybrid genes in yeast. The heterologous hybrid proteins were targeted into strongly proliferated membranes, as shown by electron microscopic and immunofluorescent analysis. Fusion proteins comprising the whole CYP1A1 polypeptide (HMG2/1-CYP1A1) exhibited 7-ethoxyresorufin-O-deethylase activity, whereas fusion proteins lacking the N-terminal 56 amino acids of CYP1A1 (HMG2/1-delta CYP1A1) were inactive and appeared to be unable to incorporate protoheme. Similar amounts of heterologous protein were detected in cells expressing HMG2/1-CYP1A1, HMG2/1-delta CYP1A1 and CYP1A1, respectively. Replacement of the N-terminal membrane anchor domain of human NADPH-cytochrome P450 oxidoreductase by the HMG2/1 peptide also resulted in a functional fusion enzyme, which was able to interact with HMG2/1-CYP1A1 and the yeast endogenous P450 enzyme lanosterol-14 alpha-demethylase.

Functional expression of fused enzymes between human cytochrome P4501A1 and human NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae.

The activity of human cytochrome P450 enzymes heterologously expressed in Saccaromyces cerevisiae cells is limited by the yeast endogenous cytochrome P450 oxidoreductase (yOR). To overcome these limitations, we constructed hybrids between human P4501A1 (CYP1A1) and human P450 oxidoreductase (hOR) by combining the cDNA encoding hOR with the CYP1A1 cDNA. In addition, in one construct, the amino terminus of hOR was replaced by the membrane anchor domain of a yeast protein. Anchoring of the fusion constructs in internal membranes either by the amino terminus of hOR or by the yeast peptide resulted in functional hybrid proteins, which were present in similar amounts as the authentic CYP1A1 in microsomal fractions of recombinant cells. Saccharomyces cerevisiae cells transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-CYP1A immunoglobulin (Ig) and anti-oxidoreductase Ig. Saccharomyces cerevisiae yOR-mutant (cpr1-) and wild-type (CPR1+) cells containing the fused enzymes exhibited CYP1A1-specific 7-ethoxyresorufin-O-deethylase activities. Reduced CO-difference spectra of microsomal fractions containing the fused enzymes indicated a proper incorporation of protoheme into the CYP1A1 domains. These results show that the chimeric proteins represent catalytically self-sufficient monooxygenase systems. The hOR domains of the hybrid proteins were also functional as cytochrome c reductases and able to activate the yeast P450 enzyme lanosterol-14 alpha-demethylase, indicating correct insertion of the chimeric proteins in internal membranes.

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

Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the loss of the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors. To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolizes two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines. This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH-CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene. Microsomes isolated from the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the Ames test. Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination. These results are discussed in light of possible mechanisms that are involved in aflatoxin B1-mediated hepatocarcinogenesis. Similarly, benzo[a]pyrene-trans-7,8-dihydrodiol and 3-amino-1-methyl-5H-pyrido[4,3-b]indole were activated to recombinagenic products, whereas benzo[a]pyrene and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline were negative in this assay. Our results argue that the constructed yeast strains may be a valuable tool for the investigation of drug-induced mitotic recombination.

The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily.

Several pleiotropic drug sensitivities have been described in yeast. Some involve the loss of putative drug efflux pumps analogous to mammalian P-glycoproteins, others are caused by defects in sterol synthesis resulting in higher plasma membrane permeability. We have constructed a Saccharomyces cerevisiae strain that exhibits a strong crystal violet-sensitive phenotype. By selecting cells of the supersensitive strain for normal sensitivity after transformation with a wild-type yeast genomic library, a complementing 10-kb DNA fragment was isolated, a 3.4-kb subfragment of which was sufficient for complementation. DNA sequence analysis revealed that the complementing fragment comprised the recently sequenced SGE1 gene, a partial multicopy suppressor of gal11 mutations. The supersensitive strain was found to be a sge1 null mutant. Overexpression of SGE1 on a high-copy-number plasmid increased the resistance of the supersensitive strain. Disruption of SGE1 in a wild-type strain increased the sensitivity of the strain. These features of the SGE1 phenotype, as well as sequence homologies of SGE1 at the amino acid level, confirm that the Sge1 protein is a member of the drug-resistance protein family within the major facilitator superfamily (MFS).

Characterization of the trp5-27 allele used to monitor drug-induced mitotic gene conversion in the Saccharomyces cerevisiae tester strain D7.

Mitotic gene conversions, among other recombinagenic events, can play an important role in the multistep process of carcinogenesis. The ability of chemicals to induce such gene conversions can easily be monitored in the Saccharomyces cerevisiae tester strain YHE2, a derivative of strain D7. For the detection of drug-induced gene conversions, two mutations in the TRP5 locus are used, trp5-12 and trp5-27. Here we report on the characterization of the stable allele trp5-27. Our analysis revealed two relevant mutations in trp5-27: (a) a transition C to T at position 121 after ATG that results in an amber stop codon and abolishes gene expression and (b) a transversion A to T at position 1555 that creates an ochre stop codon. Simultaneous amber and ochre suppression with the suppressors SUP3 and SUP11, respectively, was capable of relieving the tryptophan-requiring phenotype of strains carrying the trp5-27 allele. These findings have implications on the length of gene conversion tracts in conversion events between trp5-12 and trp5-27: conversion tracts can cover several kilobases, if the site of the mutation in trp5-12 lies outside of the positions mutated in trp5-27. Conversely, the maximal length is limited to 1435 bp, if the mutation in trp5-12 is located between the positions mutated in trp5-27.

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.