Genotyping of a gene cluster for production of colibactin and in vitro genotoxicity analysis of Escherichia coli strains obtained from the Japan Collection of Microorganisms.

INTRODUCTION: Colibactin is a small genotoxic molecule produced by enteric bacteria, including certain Escherichia coli (E. coli) strains harbored in the human large intestine. This polyketide-peptide genotoxin is considered to contribute to the development of colorectal cancer. The colibactin-producing (clb +) microorganisms possess a 54-kilobase genomic island (clb gene cluster). In the present study, to assess the distribution of the clb gene cluster, genotyping analysis was carried out among E. coli strains randomly chosen from the Japan Collection of Microorganisms, RIKEN BRC, Japan. FINDINGS: The analysis revealed that two of six strains possessed a clb gene cluster. These clb + strains JCM5263 and JCM5491 induced genotoxicity in in vitro micronucleus (MN) tests using rodent CHO AA8 cells. Since the induction level of MN by JCM5263 was high, a bacterial umu test was carried out with a cell extract of the strain, revealing that the extract had SOS-inducing potency in the umu tester bacterium. CONCLUSION: These results support the observations that the clb gene cluster is widely distributed in nature and clb + E. coli having genotoxic potencies is not rare among microorganisms.

In vitro genotoxicity analyses of colibactin-producing E. coli isolated from a Japanese colorectal cancer patient.

Colibactin is a polyketide-peptide genotoxin produced by enteric bacteria such as E. coli, and is considered to contribute to the development of colorectal cancer. We previously isolated E. coli strains from Japanese colorectal cancer patients, and in the present study we investigated the genotoxic potency of the colibactin-producing (clb+) E. coli strains that carry the polyketide synthases "pks" gene cluster (pks+) and an isogenic clb- mutant in which the colibactin-producing ability is impaired. Measurement of phosphorylated histone H2AX indicated that DNA double strand breaks were induced in mammalian CHO AA8 cells infected with the clb+ E. coli strains. Induction of DNA damage response (SOS response) by crude extract of the clb+ strains was 1.7 times higher than that of the clb- E. coli in an umu assay with a Salmonella typhimurium TA1535/pSK1002 tester strain. Micronucleus test with CHO AA8 cells revealed that infection with the clb+ strains induced genotoxicity, i.e., the frequencies of micronucleated cells infected with clb+ strain were 4-6 times higher than with the clb- strain. Since the intestinal flora are affected by dietary habits that are strongly associated with ethnicity, these data may contribute to both risk evaluation and prevention of colorectal cancer in the Japanese population.

Bioactivation mechanisms of N-hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids.

Aristolochic acids (AAs) are human nephrotoxins and carcinogens found in concoctions of Aristolochia plants used in traditional medicinal practices worldwide. Genotoxicity of AAs is associated with the formation of active species catalyzed by metabolic enzymes, the full repertoire of which is unknown. Recently, we provided evidence that sulfonation is important for bioactivation of AAs. Here, we employ Salmonella typhimurium umu tester strains expressing human N-acetyltransferases (NATs) and sulfotransferases (SULTs), to study the role of conjugation reactions in the genotoxicities of N-hydroxyaristolactams (AL-I-NOH and AL-II-NOH), metabolites of AA-I and AA-II. Both N-hydroxyaristolactams show stronger genotoxic effects in umu strains expressing human NAT1 and NAT2, than in the parent strain. Additionally, AL-I-NOH displays increased genotoxicity in strains expressing human SULT1A1 and SULT1A2, whereas AL-II-NOH shows enhanced genotoxicity in SULT1A1/2 and SULT1A3 strains. 2,6-Dichloro-4-nitrophenol, SULTs inhibitor, reduced umuC gene expression induced by N-hydroxyaristolactams in SULT1A2 strain. N-hydroxyaristolactams are also mutagenic in parent strains, suggesting that an additional mechanism(s) may contribute to their genotoxicities. Accordingly, using putative SULT substrates and inhibitors, we found that cytosols obtained from human kidney HK-2 cells activate N-hydroxyaristolactams in aristolactam-DNA adducts with the limited involvement of SULTs. Removal of low-molecular-weight reactants in the 3.5-10 kDa range inhibits the formation of aristolactam-DNA by 500-fold, which could not be prevented by the addition of cofactors for SULTs and NATs. In conclusion, our results demonstrate that the genotoxicities of N-hydroxyaristolactams depend on the cell type and involve not only sulfonation but also N,O-acetyltransfer and an additional yet unknown mechanism(s). Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Synthesis and In Vitro Biological Evaluation of Psoralen-Linked Fullerenes.

Photodynamic therapy (PDT) is a widely used medicinal treatment for the cancer therapy that utilizes the combination of a photosensitizer (PS) and light irradiation. In this study, we synthesized two novel C60 fullerene derivatives, compounds 1 and 2, with a psoralen moiety that can covalently bind to DNA molecules via cross-linking to pyrimidine under photoirradiation. Along with several fullerene derivatives, the biological properties of several novel compounds have been evaluated. Compounds 1 and 2, which have been shown to induce the production of hydroxyl radicals using several ROS detecting reagents, induced DNA strand breaks with relatively weak activities in the in vitro detection system using a supercoiled plasmid. However, the psoralen-bound fullerene with carboxyl groups (2) only showed genotoxicity in the genotoxicity assay system of the umu test. Compound 2 was also seen to have cytotoxic activities in several cancer cell lines at higher doses compared to water-soluble fullerenes.

Advanced Approaches to Model Xenobiotic Metabolism in Bacterial Genotoxicology In Vitro.

During the past 30 years there has been considerable progress in the development of bacterial test systems for use in genotoxicity testing by the stable introduction of expression vectors (cDNAs) coding for xenobiotic-metabolizing enzymes into bacterial cells. The development not only provides insights into the mechanisms of bioactivation of xenobiotic compounds but also evaluates the roles of enzymes involved in metabolic activation or inactivation in chemical carcinogenesis. This review describes recent advances in bacterial genotoxicity assays and their future prospects, with a focus on the development and application of genetically engineering bacterial cells to incorporate some of the enzymatic activities involved in the bio-activation process of xenobiotics. Various genes have been introduced into bacterial umu tester strains encoding enzymes for genotoxic bioactivation, including bacterial nitroreductase and O-acetyltransferase, human cytochrome P450 monooxygenases, rat glutathione S-transferases, and human N-acetyltransferases and sulfotransferases. Their application has provided new tools for genotoxicity assays and for studying the role of biotransformation in chemical carcinogenesis in humans.

Development and progress for three decades in umu test systems.

Umu test have been widely used to predict the detection and assessment of DNA- damaging chemicals in environmental genotoxicity field for three decades. This test system is more useful with respect to simplicity, sensitivity, rapidity, and reproducibility. A review of the literature on the development of the umu test is presented in this article. The contents of this article are included a description of numerous data using the umu test. This test have been fully evaluated and used in many directions. Different genetically engineered umu systems introducing bacterial and rat or human drug metabolizing enzymes into the umu tester strains, have been successfully established and are considered as useful tools for genotoxicity assays to study the mechanisms of biotransformation in chemical carcinogenesis. Actually, we developed that two types of bacterial metabolizing enzymes and 4 types of rat and human metabolizing enzyme DNAs are expressed in these strains such as nitroreductase and O-acetyltransferase, cytochrome P450, N-acetyltransferases, sulfotransferases, and glutathione S-transferases, respectively. Due to increasing numbers of minute environmental samples and new pharmaceuticals, a high-throughput umu test system using Salmonella typhimurium TA1535/pSK1002, NM2009, and NM3009 strains provides a useful for these genotoxicity screening. I also briefly describe the first attempts to incorporate such umu tester strain into photo-genotoxicity test.

Evaluation of genotoxic effects of surface waters using a battery of bioassays indicating different mode of action.

With the burgeoning contamination of surface waters threatening human health, the genotoxic effects of surface waters have received much attention. Because mutagenic and carcinogenic compounds in water cause tumors by different mechanisms, a battery of bioassays that each indicate a different mode of action (MOA) is required to evaluate the genotoxic effects of contaminants in water samples. In this study, 15 water samples from two source water reservoirs and surrounding rivers in Shijiazhuang city of China were evaluated for genotoxic effects. Target chemical analyses of 14 genotoxic pollutants were performed according to the Environmental quality standards for surface water of China. Then, the in vitro cytokinesis-block micronucleus (CBMN) assay, based on a high-content screening technique, was used to detect the effect of chromosome damage. The SOS/umu test using strain TA1535/pSK1002 was used to detect effects on SOS repair of gene expression. Additionally, two other strains, NM2009 and NM3009, which are highly sensitive to aromatic amines and nitroarenes, respectively, were used in the SOS/umu test to avoid false negative results. In the water samples, only two of the genotoxic chemicals listed in the water standards were detected in a few samples, with concentrations that were below water quality standards. However, positive results for the CBMN assay were observed in two river samples, and positive results for the induction of umuC gene expression in TA1535/pSK1002 were observed in seven river samples. Moreover, positive results were observed for NM2009 with S9 and NM3009 without S9 in some samples that had negative results using the strain TA1535/pSK1002. Based on the results with NM2009 and NM3009, some unknown or undetected aromatic amines and nitroarenes were likely in the source water reservoirs and the surrounding rivers. Furthermore, these compounds were most likely the causative pollutants for the genotoxic effect of these water samples. Therefore, to identify causative pollutants with harmful biological effects, chemical analyses for the pollutants listed in water quality standards is not sufficient, and single-endpoint bioassays may underestimate adverse effects. Thus, a battery of bioassays based on different MOAs is required for the comprehensive detection of harmful biological effects. In conclusion, for genotoxicity screening of surface waters, the SOS/umu test system by using different strains combined with the CBMN assay was a useful approach.

Roles of human sulfotransferases in genotoxicity of carcinogens using genetically engineered umu test strains.

Human sulfotransferase (SULT) 1A1, 1A2, and 1A3 cDNA genes were subcloned separately into the pTrc99A(KM) vector. The generated plasmids were introduced into the Salmonella typhimurium O-acetyltransferase-deficient strain NM6000 (TA1538/1,8-DNP/pSK1002), resulting in the new strains NM7001, NM7002, and NM7003. We compared the sensitivities of these three strains with the parental strain NM7000 against 51 chemicals including aromatic amines, nitroarenes, alkenylbenzenes, estrogens-like chemicals, and other compounds with and without S9 mix by making use of the umu test system that is based on the bacterial SOS induction. 2-Amino-6-methyl-dipyrido[1,2-α:3',2'-d]imidazole, 3-methoxy-4-aminoazobenzene, 3-nitrobenzanthrone, 5-nitroacenaphthene, and 3,9-dinitrofluoranthene caused high genotoxicity in the NM7001 strain. The genotoxic effects of 2-aminofluorene, 2-acetylaminofluorene, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-nitrofluorene, 1-nitropyrene, and 2-nitropropane were stronger in the NM7002 strain compared with the NM7001 and NM7003 strains. Among the tested benzylic and allylic compounds, 1-hydroxymethylpyrene was detected in the NM7001 strain with the highest sensitivity. Estragole and 1'-hydroxysafrole exhibited strong genotoxicity in the NM7003 strain. The estrogen-like chemicals such as bisphenol A, genistein, p,n-nonylphenol, and 4-hydroxytamoxifen were not detected as genotoxins in any strain used. Collectively, the present results suggest that the generated test strains are valuable tools in order to elucidate the role of SULT enzymes in the bioactivation of chemicals to environmental carcinogens.

Antigenotoxic activity of naturally occurring furanocoumarins.

This study was designed to investigate the antigenotoxic effects of a series of naturally occurring furanocoumarins (NOFs) including isoimperatorin, imperatorin, (+)-oxypeucedanin, (+)-byakangelicol, and (+)-byakangelicine on antigenotoxic activities against genotoxicity induced by carcinogens [furylfuramide and N-methyl-N'-nitro-N-nitrosoguanidine], and procarcinogens 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) and 2-amino-3,4-dimethyl-3H-imidazo-[4,5-f] quinoline (MeIQ)] to genotoxic metabolites catalyzed by rat S9 or rat and human recombinant cytochrome P450 (CYP) 1As by using the umu test based on SOS response. Five different NOFs, which were found in the human diets, strongly inhibited the umuC induction by procarcinogens, but did not be affected by carcinogens. Notably, isoimperatorin and (+)-byakangelicol were found to be potent inhibitors on the metabolic activation of PBTA-4 and MeIQ to genotoxic metabolites catalyzed by rat and human CYP1A1, or rat and human CYP1A2, respectively. In addition, to elucidate the mechanism of their antigenotoxic effects against procarcinogens, the effects of NOFs on rat and human CYP1A1- or rat and human CYP1A2-related enzyme activities of 7-ethoxyresorufin-O-deethylase (EROD) were also investigated. Reduction of the EROD activities by some of the NOFs with IC(50) values of 0.23-20.64 µM was found to be due to strong inhibition of CYP1A1 and CYP1A2 dependent monooxygenases. Furthermore, the mechanism of inhibitions by NOFs on human CYP1A1 and CYP1A2 was analyzed by means of Dixon plots plus Cornish-Bowden plots. The kinetic studies of inhibition types revealed that these compounds inhibited the human CYP1A1 and CYP1A2 a variety of modes rather than by a uniform one. Moreover, experiments with a two-stage incubation indicated that NOFs, except for imperatorin, inhibited human CYP1A1 in a mechanism-based manner, but directly inhibited human CYP1A2. This data suggest that certain NOFs, to which humans are exposed in the diet, may be capable of affecting the metabolic activation of procarcinogens due to inhibitions of CYP1A1 and CYP1A2 enzymes.

The novel assay method for nicotine metabolism to cotinine using high performance liquid chromatography.

Nicotine is the primary psychoactive component in tobacco. It is taken into the body by tobacco smoking, and mainly metabolized to cotinine in the hepatic cytochrme P450 (CYP) 2A6. The objective of this study was to develop a sensitive method for the determination of nicotine metabolism to cotinine using HPLC. The internal standard, trans-4'-carboxycotinine methyl ester was synthesized with a simple method. The nicotine and cotinine were separated completely and detected by C(18) 5-µm analytical column (L-column Octa decyl silyl (ODS), 150 mm × 4.6 mm i.d.) equipped with a C(18) 5-µm guard column (L-column ODS, 10 mm × 4.6 mm i.d.) and ultraviolet detection at 260 nm. The detection limit of the assay was 0.05 µM for cotinine (n=5, R.S.D) and 0.1 µM for nicotine. Thus the present results provided a sensitive and useful method for the determination of nicotine metabolism catalyzed by CYP2A6.

Genotoxicity of acrylamide in vitro: Acrylamide is not metabolically activated in standard in vitro systems.

The recent finding that acrylamide (AA), a genotoxic rodent carcinogen, is formed during the frying or baking of a variety of foods raises human health concerns. AA is known to be metabolized by cytochrome P450 2E1 (CYP2E1) to glycidamide (GA), which is responsible for AA's in vivo genotoxicity and probable carcinogenicity. In in-vitro mammalian cell tests, however, AA genotoxicity is not enhanced by rat liver S9 or a human liver microsomal fraction. In an attempt to demonstrate the in vitro expression of AA genotoxicity, we employed Salmonella strains and human cell lines that overexpress human CYP2E1. In the umu test, however, AA was not genotoxic in the CYP2E1-expressing Salmonella strain or its parental strain. Moreover, a transgenic human lymphoblastoid cell line overexpressing CYP2E1 (h2E1v2) and its parental cell line (AHH-1) both showed equally weak cytotoxic and genotoxic responses to high (>1 mM) AA concentrations. The DNA adduct N7-GA-Gua, which is detected in liver following AA treatment in vivo, was not substantially formed in the in vitro system. These results indicate that AA was not metabolically activated to GA in vitro. Thus, AA is not relevantly genotoxic in vitro, although its in vivo genotoxicity was clearly demonstrated.

Genotoxicity of 3,6-dinitrobenzo[e]pyrene, a novel mutagen in ambient air and surface soil, in mammalian cells in vitro and in vivo.

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP), newly identified in airborne particles and surface soil, is a potent mutagen in Salmonella typhimurium. The present study investigated the genotoxic potency of 3,6-DNBeP in vitro and in vivo using mammalian cell strains (Chinese hamster CHL/IU and human HepG2) and ICR mice, respectively. In the hprt gene mutation assay using HepG2 cells, the spontaneous mutant frequency was 61.1 per 10(5) clonable cells, which increased to 229 per 10(5) clonable cells after treatment with 1.0 microg/ml (3 microM) 3,6-DNBeP. Notably, in HepG2 cells with increased N-acetyltransferase 2 activity, the mutant frequency increased to 648 per 10(5) clonable cells by treatment of 1.0 microg/ml (3 microM) 3,6-DNBeP. The sister chromatid exchange frequency increased approximately three times the control level in HepG2 cells treated with 3,6-DNBeP at a concentration of 1.0 microg/ml (3 microM). In HepG2 and CHL/IU cells, the frequency of the cells with micronuclei was 0.9 and 1.2%, and the frequencies increased to 2.3 and 7.6% after 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment, respectively. The H2AX phosphorylation level increased 8-fold compared with the background level with 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment in HepG2 cells. Moreover, the comet assay showed that 3,6-DNBeP produced DNA damage in the cells of liver, kidney, lung and bone marrow in ICR mice 3 h after intraperitoneal injection at 40 mg/kg (0.12 mmol/kg) body weight. These data indicate that 3,6-DNBeP is genotoxic to mammalian cells in vitro and in vivo.

Activation of aminophenylnorharman, aminomethylphenylnorharman and aminophenylharman to genotoxic metabolites by human N-acetyltransferases and cytochrome P450 enzymes expressed in Salmonella typhimurium umu tester strains.

Norharman (9H-pyrido[3,4-b]indole) and harman (1-methyl-9H-pyrido[3,4-b]indole) contained in cigarette smoke and cooked foodstuffs, are non-mutagenic to Salmonella strains, but show co-mutagenicity with S9 mixture in the presence of aniline or o-toluidine. The resulting 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (aminomethylphenylnorharman, AMPNH) and 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole (aminophenylharman, APH) are produced by coupling of norharman and aniline, norharman and o-toluidine, and harman and aniline in the presence of S9 mixture, respectively. To clarify the role of human cytochrome P450 (P450) and N-acetyltransferase (NAT) enzymes in the metabolic activation of APNH, AMPNH and APH, we determined the genotoxicity of these coupling chemicals using a variety of umu tester strains established in our laboratories. APNH, AMPNH and APH induced umuC gene expression more strongly in a bacterial O-acetyltransferase-overproducing strain than the parent strain. These chemicals were also found to induce umuC gene expression in NAT2-overexpressing strain at much higher rate than the NAT1-overexpressing strain. Among seven OY strains expressing human P450s and NADPH-P450 reductase used, the genotoxicity of APNH, AMPNH and APH was detected in OY1002/1A2 strain, OY1002/1A1 and OY1002/1A2 strains, and in OY1002/1A2 strain, respectively. From these results, it is concluded that APNH, AMPNH and APH are mainly bioactivated by P450 1A2 and NAT2, followed by NAT1 enzymes. P450 1A1 was also found to activate AMPNH at relatively slower rates.

Suppression of MeIQ-induced SOS response by allylbenzenes from Asiasarum heterotropoides in the Salmonella typhimurium OY1001/1A2 umu test.

Three allylbenzenes from Asiasarum heterotropoides, methyleugenol (1), elemicin (2) and gamma-asaron (3) showed suppressive effects on umu gene expression of the SOS response in the Salmonella typhimurium OY1001/1A2 umu test against the mutagen 2-amino-3,4-dimethylimidazo[4,5-f ]quinoline (MeIQ). Gene expression was suppressed 70.0, 75.9 and 81.7% at a concentration of 0.4 mM, respectively. The ID50 values (50% inhibition dose) of these compounds were 0.125, 0.098 and 0.059 mM, respectively. On the other hand, compounds 1-3 showed weak suppressive effects of the SOS-inducing activity on activated MeIQ.

Analysis of the involvement of human N-acetyltransferase 1 in the genotoxic activation of bladder carcinogenic arylamines using a SOS/umu assay system.

Human acetyltransferase genes NAT1 or NAT2 were expressed in a Salmonella typhimurium strain used to detect the genotoxicity of bladder carcinogens. To clarify whether the human and rodent bladder carcinogenic arylamines are activated via either NAT1 or NAT2 to cause genotoxicity, a SOS/umu genotoxicity assay was used, with the strains S. typhimurium NM6001 (NAT1-overexpressing strain), S. typhimurium NM6002 (NAT2-overexpressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain). Genotoxicity was measured by induction of SOS/umuC gene expression in the system, which contained both an umuC"lacZ fusion gene and NAT1 or NAT2 plasmids. 4-Aminobiphenyl, 2-acetylaminofluorene, beta-naphthylamine, o-tolidine, o-anisidine, and benzidine exhibited dose-dependent induction of the umuC gene in strain NM6001. Although the induction of umuC by these chemicals was observed in the NM6002 strain, the induction was considerably lower than in the NM6001 strain. In the parent strain, NM6000, none of these compounds induced umuC gene expression. We also determined activation of these chemicals by recombinant human cytochrome P450 (P450 or CYP) 1A2 enzyme in three S. typhimurium tester strains. The activation of the chemicals was stronger in the NM6001 strain than that in NM6002. The specific NAT1 inhibitor 5-iodosalicylic acid inhibited umuC gene expression induced by aromatic amines used. These results could provide evidence that the bladder carcinogenic aromatic amines are mainly activated by the NAT1 enzyme to produce DNA damage rather than NAT2. The NAT1-overexpressing strain can be used to determine the genotoxic activation of bladder carcinogenic arylamines in the umu test and could provide a tool for predicting the carcinogenic potential of arylamines.

Use of a high-throughput umu-microplate test system for rapid detection of genotoxicity produced by mutagenic carcinogens and airborne particulate matter.

In the present study, we developed a rapid umu-microplate test system that uses the nitroreductase- and O-acetyltransferase-overproducing Salmonella typhimurium strain NM3009 and the O-acetyltransferase-overproducing S. typhimurium strain NM2009 to detect genotoxic activity in small volume samples. The assay was used to test the genotoxicity of several standard mutagens and environmental samples. Exponentially growing cultures of NM3009, NM2009, and the parental strain TA1535/pSK1002 were incubated in 96-well microplates with test chemicals both in the presence and in the absence of rat liver S9. The relative beta-galactosidase activities were then determined colorimetrically using either chlorophenol red-beta-D-galactopyranoside (CPRG) or O-nitrophenyl-beta-D-galactopyranoside (ONPG) as a measure of umuC gene induction activity. The sensitivities of NM3009 without S9 mix and NM2009 with S9 mix to nitroarenes and aromatic amines were up to 24- to 75-fold higher than those of the parent strain. Induction of umuC gene expression was detected more readily with CPRG than ONPG. The umu-microplate assay also detected genotoxicity in organic extracts of particulate matter from air samples collected in Osaka City, Japan. The pattern of the responses suggested that the genotoxic activity of the particulate extract was due primarily to nitrated polycyclic aromatic hydrocarbons. Our results indicate that the umu-microplate assay may be a useful way of carrying out rapid screens for genotoxicity in small-volume environmental samples.

Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays.

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity.