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.

Insight into the generation of toxic products during chloramination of carbamazepine: Kinetics, transformation pathway and toxicity.

As a widely used antiepileptic drug, carbamazepine (CBZ) has been frequently detected in aquatic environments, even in drinking water. Chloramine is a widely used alternative disinfectant due to its low-level formation of regulated disinfection byproducts (DBPs). However, there is previous evidence linking product mixtures of chloraminated CBZ to stronger DNA damage effects than those caused by CBZ itself. The present study further investigated the reaction rate, transformation mechanism and multi-endpoint toxicity of transformation products (TPs) of CBZ treated with NH2Cl under different pH conditions. The results showed that the reaction between CBZ and NH2Cl at pH 8.5, where NH2Cl is stable, is a second-order reaction with a rate of 4.2 M-1 h-1. Compared to both alkaline and acidic conditions, CBZ was quickly degraded at pH 7. This indicated that HOCl produced from NH2Cl hydrolysis is more effective in degrading CBZ than NH2Cl and NHCl2. Furthermore, the concentration variation of four TPs formed during the chloramination of CBZ under different pH conditions was investigate by quantitative analysis, and the transformation pathway from CBZ to 9(10H)-acridone was confirmed. Three of the detected TPs showed cytotoxicity, DNA damage effects or chromosome damage effects. Acridine and 9(10H)-acridone, which accumulated with increasing time, showed higher cytotoxic or genotoxic effects than CBZ itself. In addition, a similar transformation mechanism was observed in real ambient water during simulated chloramination with a low level of CBZ. These results suggested that despite the chloramination of CBZ being slower than chlorination, TPs with higher cytotoxicity or genotoxicity may lead to greater toxic risks.

Chlorination, chloramination and ozonation of carbamazepine enhance cytotoxicity and genotoxicity: Multi-endpoint evaluation and identification of its genotoxic transformation products.

Investigations have focused on the removal and transformation of pharmaceuticals during drinking water and wastewater treatment. In the present study, we investigated for the first time the changes of the cytotoxicity and genotoxicity based on different modes of action (MoAs) during chlorination, chloramination and ozonation processes of the anti-epileptic drug carbamazepine (CBZ). The results illustrated that ozonation enhanced the cytotoxicity and the chromosome damage effects on CHO-K1 cells detected by cytokinesis-block micronucleus (CBMN) assay based on high-content screening technique, though ozonation showed the highest removal efficiency for CBZ. Non-target chemical analysis followed by quantitative structure-activity relationship (QSAR) analysis for the transformation products (TPs) suggested that the chromosomal damage effects could probably be attributed to 1-(2-benzaldehyde)-4-hydro-(1H,3H)-quinazoline-2-one (BQM) and 1-(2-benzaldehyde)-(1H,3H)-quinazoline-2,4-dione (BQD). In contrast to CBZ itself and the ozonated sample, the chlorinated and chloraminated samples caused DNA damage effects in SOS/umu test. Acridine, 9 (10) H-acridone, chlorinated 9 (10) H-acridone and TP-237, which were first identified in the chlorination or chloramination processes, were predicted to be the DNA damaging agents. These genotoxic TPs were primarily generated from the oxidation of seven-membered N-heterocyclic in CBZ. This study highlighted the potential adverse effects generated in ozonation process and the oxidation of N-heterocyclic containing pollutants.

Development of a high-throughput genotoxicity assay using Umu test strains expressing human cytochrome P450s and NADPH-P450 reductase and bacterial O-acetyltransferase.

Umu test is one of the in vitro genotoxicity test that has been used widely. It was developed as a high-throughput test system using the 96-well microplate. We have previously constructed new umu test strains for the evaluation of genotoxicity of procarcinogenic metabolic products formed by cytochrome P450 (CYP) enzymes. In this study, a highly sensitive high-throughput genotoxicity test was developed using four umu test strains (OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4) that express human CYPs and NADPH-P450 reductase. We found that the modified umu-microplate method was more sensitive than the conventional microplate method using strain OY1002/1A2. In addition, the new microplate method was better able to detect genotoxicity than the test tube method when the strain OY1002/1A2 was used and had similar sensitivity for the remaining three strains. When the microplate method was used, OY1002/1A2 showed stronger umuC gene expression in the presence of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-aminofluorene, and 2-aminoanthracene compared to other strains. We also confirmed CYP1A2 expression in OY1002/1A2 in this condition. These results indicate that the microplate version of this test system can detect the genotoxicity of heterocyclic and aromatic amines with high sensitivity and can be used for high-throughput screening of potentially genotoxic compounds. Environ. Mol. Mutagen. 58:209-216, 2017. © 2017 Wiley Periodicals, Inc.

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.

Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

Use of a new enzyme extraction system to improve the sensitivity of SOS/umu test and application to environmental samples.

The purpose of this study was to find a better enzyme extraction reagent for the SOS/umu test to replace the conventional one (the combination of sodium dodecyl sulfate (SDS) and Z-buffer), which has the disadvantage of denaturing ß-galactosidase leading to decreased measurement sensitivity. By adopting a microplate system, the performance of the umu test using BugBuster Master Mix, a commercially available enzyme extraction reagent, was compared with that using the conventional reagent for detecting the genotoxicity of known mutagens as well as environmental samples. BugBuster Master Mix was found to increase the detection sensitivities of the selected genotoxins and environmental water samples, due to the fact that it doesn't denature ß-galactosidase. The result of this study showed that BugBuster Master Mix could be a better enzyme extraction reagent for umu test.

Contributions of flumequine and nitroarenes to the genotoxicity of river and ground waters.

The SOS/umuC assay was performed in conjunction with analytical measurements to identify potential genotoxins in river and adjacent ground waters in the Jialu River basin, China. The major genotoxic activities of the river and adjacent ground waters occurred in the same two fractions (F4 and F11) when assayed using the Salmonella typhimurium strain TA1535/pSK1002. This indicates that ground water near the Jialu River was influenced by the river water. LC-MS/MS analysis indicated that flumequine accounted for 86% and 76% of the genotoxicity in fraction F11 of the river and adjacent ground waters, respectively. When HPLC fractions were tested using the strain NM3009, three fractions showed genotoxic activities for river water sample, while no fractions from ground water samples elicited genotoxic activities. The specific response to the strain NM3009 in one fraction compared with the strain TA1535/pSK1002 suggested the presence of nitroarenes. However, we failed to identify the exact nitroarenes when GC-MS analysis was used to analyze nitroarenes which are well detected in air and soil samples in previous papers.

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.

Evaluation of photo-genotoxicity using the umu test in strains with a high sensitivity to oxidative DNA damage.

Six compounds known to be photo-genotoxic were assayed with the photo-irradiated 96-well umu test system using the original Salmonella strain TA1535/pSK1002 as well as the newly developed strains NM8001 and NM8021. The latter two strains were obtained by introducing the pSK1002 plasmid into strains YG3001 and YG3021. These strains are highly sensitive to oxidative DNA damage owing to the deletion of the nucleotide excision repair enzyme uvrB and the base excision repair enzyme mutY and the nucleotide excision repair enzyme uvrB and the base excision repair enzymes mutY and mutM, respectively. Among the compounds tested under UVA irradiation, methylene blue, neutral red and dichlorobenzidine showed only a slight induction of ß-galactosidase activity, whereas 8-methoxypsoralen, chloropromazine and 9,10-dimethylbenzanthracene showed a significant increase in the relative LacZ level as an indicator of genotoxicity. The activity of NM8001 induced by the photo-genotoxins was quite similar to that of NM8021, which indicated that the deficiency of mutY did not affect detection of the selected photo-genotoxins. With NM8001, under UVA light, riboflavin was able to induce the SOS response in bacterial cells. Moreover, neutral red was also found to exert photo-genotoxicity under fluorescent light. Phenalenone, a known atmospheric contaminant present in large amounts, showed positive response with NM8001 under UVA. These results indicate that the photo-irradiated 96-well version of the umu test can be used for rapid screening of the photo-genotoxicity of compounds.

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.

Suppression of SOS-inducing activity of chemical mutagens by metabolites from microbial transformation of (-)-isolongifolene.

In this study, biotransformation of (-)-isolongifolene (1) by Glomerella cingulata and suppressive effect on umuC gene expression by chemical mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) and aflatoxin B(1) (AFB(1)) of the SOS response in Salmonella typhimurium TA1535/pSK1002 were investigated. Initially, 1 was carried out the microbial transformation by G. cingulata. The result found that 1 was converted into (-)-isolongifolen-9-one (2), (-)-(2S)-13-hydroxy-isolongifolen-9-one (3), and (-)-(4R)-4-hydroxy-isolongifolen-9-one (4) by G. cingulata, and their conversion rates were 60, 25, and 15%, respectively. The metabolites suppressed the SOS-inducing activity of furylfuramid and AFB(1) in the umu test. Comound 2 showed gene expression by chemical mutagens furylfuramide and AFB(1) was suppressed 54 and 50% at <0.5 mM, respectively. Compound 2 is the most effective compound in this experiment.

Genotoxic activation of the environmental pollutant 3,6-dinitrobenzo[e]pyrene in Salmonella typhimurium umu strains expressing human cytochrome P450 and N-acetyltransferase.

3,6-Dinitrobenzo[e]pyrene (DNBeP) is a potent mutagen identified in surface soil in two metropolitan areas of Japan. We investigated whether DNBeP can cause genotoxicity through any metabolic activation pathway in bacteria using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. To further clarify the role of human cytochrome P450 (P450 or CYP) and N-acetyltransferase (NAT) enzymes in the bioactivation of DNBeP to genotoxic metabolites, we determined the genotoxicity of DNBeP using a variety of umu tester strains expressing human P450 and NAT enzymes. The dose-dependent induction of umuC by DNBeP was observed at concentrations between 0.01 and 1nM in the O-AT-expression strain, but not in the O-AT-deficient strain. In the CYP3A4-, CYP1A2-, CYP1A1-, and CYP1B1-expressing strains, DNBeP was found to be activated to reactive metabolites that cause the induction of umuC gene expression compared with the parent strain. The induction of DNBeP in the NAT2-expressing strain had a 10-fold lower concentration than that in the NAT1-expressing strain. Collectively, these results suggest that nitroreduction by human CYP1A2, CYP3A4, and CYP1A1 and O-acetylation by human NAT2 contributed to the genotoxic activation of DNBeP to its metabolites.