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Mutagenesis is an important technique for microbial mutation breeding. As the source of mutations, DNA damage extent is a key indicator for the effectiveness of mutagenesis. Therefore, a rapid and easy DNA damage quantification method is required for the comparison of mutagenesis effects and development of mutagenesis tools. Here, we used the umu-microplate test system to quantitatively compare the DNA damage strength caused by Atmospheric and Room-Temperature Plasma (ARTP) and other traditional mutagenesis methods including: ultraviolet radiation (UV), diethyl sulfate (DES) and 4-Nitroquinoline-1-oxide (4-NQO). The test strain of Salmonella typhimurium TA1535/pSK1002 was used to monitor the time-course profile of β-galactosidase activity induced by DNA damage caused by different mutagenesis methods using a microplate reader. The umu-microplate test results showed that ARTP caused higher extent of DNA damage than UV and chemical mutagens, which agrees well with the result obtained by SOS-FACS-based quantification method as reported previously. This umu-microplate test is accessible for broad researchers who are lack of the expensive FACS instruments and allows the quick quantitative evaluation of DNA damage among living cells for different mutagenesis methods in the study of the microbial mutation breeding. © 2020 The Chemical Industry and Engineering Society of China, and Chemical Industry Press Co., Ltd.. All rights reserved.

The genotoxic activities of effluents from drainage water treatment plants were examined by using the novel umu tester strain NM8001, which lacks MutMst genes. To enhance the sensitivity of the LacZ assay, a BugBuster mix protein extraction reagent and TokyoGreen-β Gal for a fluorescence-galactosidase substrate were applied. Of the 24 sampling locations present in Kanagawa prefecture, Japan, water extracts from nine sampling points showed apparent genotoxic activities without metabolic activation. In contrast, water extracts from the upper sites of these water treatment plants did not show any significant genotoxic activities. The selected samples with genotoxic activity did not show significant mutagenicity toward Ames strains TA98 and TA100. Genotoxicity was also well correlated with the activity of a classical umu strain of TA1535/pSK1002; these findings indicate that the genotoxicity induced by oxidative damage was not a significant component of the genotoxicity.

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.

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.

application/pdf, Genes and Environment. 2020, 42,(Article number: 12), P.1-6

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.

A surface-roughened clad (S-clad) current collector significantly extends the cycle-life of the lithium ion negative electrode composed of a silicon micropowder and an aqueous binder. The high tensile strength of the S-clad is also proved to be important for improving the battery performance of the electrode by comparison to a surface-roughened pure Cu current collector. Moreover, adding 10 vol.% fluoroethylene carbonate to the electrolyte further extends the cycle-life of the Si electrode. The synergic effect of the high adhesive and tensile strength of the S-clad current collector as well as the electrolyte additive results in maintaining the reversible capacity of 1000 mA h g−1 for more than 1000 cycles, in which 1.0–1.2 mg cm−2 of the active material is loaded on the electrode.

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

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.

We develop a clad foil current collector with a high tensile strength that endures a large volume change in the active material during the charge and discharge, such as the Si-based materials. The nano-Si negative electrode with the clad current collector retains 76% of the initial capacity after 40 cycles, while the capacity of the nano-Si electrode with a conventional Cu foil drops to less than 70% only after 10 cycles. A full cell with the SiO negative electrode and the LiFePO4 positive electrode retains more than 90% of its capacity at the 10th cycle after 800 cycles. The conventional rolled Cu foil wrinkles during the cycling test. The high-strength clad current foil hardly deforms during the test regardless of the electrode size.

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.

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.

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.

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.

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.

Human sulfotransferase (SULT) 1A1, 1A2, and 1A3 cDNA genes were subcloned separately into the pTrc99AKM 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-a: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. Environ. Mol. Mutagen. 2012. (c) 2011 Wiley Periodicals, Inc.

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 IC50 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. Environ. Mol. Mutagen., 2011. © 2011 Wiley-Liss, Inc.

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% at0.5 mM, respectively. Compound 2 is the most effective compound in this experiment.

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N -acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O -acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O -acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant ( Trichoplusia ni ) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. α-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 ( O -AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O -acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.

3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic compound identified in diesel exhaust, airborne particulate matter, soil, and water. To address whether 3-NBA shows genotoxic effects through any metabolic activation pathway, we determined the genotoxicity of 3-NBA 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. In order to further clarify the role of human cytochrome P450 (P450 or CYP) enzyme in the bioactivation of 3-NBA to genotoxic metabolites, umu tester strains S. typhimurium OY1022/1A1, OY1022/1A2, OY1022/1B1, and OY1022/3A4, which express four respective human cytochrome P450 enzymes, NADPH-cytochrome P450 reductases (NPR) and O-AT were established by introducing two plasmids into S. typhimurium TA1535NR/1,8-DNP (nitroreductase-deficient and resistant to 1,8-dinitropyrene, probably due to functional loss of O-AT), one carrying both P450 and reductase cDNA in a biocistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC'-'lacZ fusion genes. Induction of umuC gene expression could be monitored by measuring the cellular β-galactosidase activity produced by the umuC'-'lacZ fusion gene. Although the induction of umuC by 3-NBA was weak in TA1535/pSK1002, NM1000, and NM2000 strains, the induction was considerably potent in NM2009 and NM3009 strains. 3-NBA was also found to induce umuC gene expression in OY1022/1A1 and OY1022/3A4 strains and, to a lesser extent, OY1022/1A2 and OY1022/1B1 strains, at a much higher rate than the parental OY1022/pCW strain. Collectively, these results suggest nitroreduction and O-acetylation by bacterial NR and O-AT, respectively, and nitroreduction by human CYP1A1, 3A4, 1A2, and 1B1 and NPR could contribute to the genotoxic activation of 3-NBA to produce reactive metabolites.

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.

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.

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, β-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.

The synthesis of the NaxCo2O4 with a layered crystal structure was attempted by using the polymerized complex (PC) method or citric acid complex (CAC) method, both of which are a kind of chemical solution process. The effects of these chemical solution processes on the crystallographic anisotropy, microstructure and thermoelectric properties of the sintered NaxCo2O4 were investigated and compared to those of the conventional SSR method. In the case of the PC method, the sintered body with higher chemical homogeneity and finer microstructure than those of the SSR sample was obtained, resulting in the high thermoelectric performance. However, the crystallographic orientation was almost the same as that of the SSR sample. On the other hand, in the case of the CAC method, a flaky powder precursor was formed by grain growth of the NaxCo2O4 phase along the c-plane. The NaxCo2O4 sample sintered from this powder precursor showed a high crystallographic anisotropy and good chemical homogeneity, so the CAC process was found to be quite effective for synthesizing a crystallographically oriented NaxCo2O4 polycrystal with high thermoelectric performance.

The number, spatial distribution, and significance of genetically distinguishable ecotypes of prokaryotes in the environment are poorly understood. Oda et al. (Y. Oda, B. Star, L. A. Huisman, J. C. Gottschal, and L. J. Forney, Appl. Environ. Microbiol. 69:xxx-xxx, 2003) have shown that Rhodopseudomonas palustris ecotypes were lognormally distributed along a 10-m transect and that multiple strains of the species could coexist in 0.5-g sediment samples. To extend these observations, we investigated the clonal diversity of R. palustris in 0.5-g samples taken from the corners and center of a 1-m square. A total of 35 or 36 clones were recovered by direct plating from each sample and were characterized by BOX A1R repetitive element-PCR genomic DNA fingerprinting. Isolates with fingerprint images that were ≥80% similar to each other were defined as the same genotype. Among the 178 isolates studied, 32 genotypes were identified, and each genotype contained between 1 and 40 isolates. These clusters were consistent with minor variations found in 16S rRNA gene sequences. The Shannon indices of the genotypic diversity within each location ranged from 1.08 (5 genotypes) to 2.18 (13 genotypes). Comparison of the rank abundance of genotypes found in pairs of locations showed that strains from three locations were similar to each other, with Morisita-Horn similarity coefficients ranging from 0.59 to 0.71. All comparisons involving the remaining two locations resulted in coefficients between 0 and 0.12. From these results we inferred that the patterns of ecotype diversity at the sampling site are patchy at a 1-m scale and postulated that factors such as mixing, competitive interactions, and microhabitat variability are likely to be responsible for the maintenance of the similarities between some locations and the differences between others.

The citric acid complex (CAC) process and subsequent pressureless sintering were explored towards the synthesis of a c-axis oriented NaxCo2O4 thermoelectric polycrystal. The effects of the CAC process on the shape of powder precursor, crystallographic anisotropy of a sintered compact and its thermoelectric properties were investigated. The CAC process provided a plate-like powder precursor composed of the γ-NaxCo2O4 phase, reflecting its layered crystal structure. During compacting and sintering of this powder, the plate-like grains were well aligned along the pressed plane because of their specific shape, resulting in the sintered body with large crystallographic anisotropy. The CAC method was also effective for synthesizing the sample with compositional homogeneity and fine microstructure. The electrical resistivity of the CAC sample was almost the same as that of the sample prepared by the conventional solid state reaction method in spite of the fine microstructure, because of its high degree of c-axis orientation. The Seebeck coefficient was significantly improved by adopting the CAC method, resulting in the large enhancement of the power factor.

Layered P2 type Na x Co 2 O 4 thermoelectric oxides partially substituted by 3d transition metal (up to 15 at%: Cr, Mn, Fe, Ni, or Zn) for Co were synthesized through calcination and sintering in air. The Cr-substituted samples were mostly decomposed into the NaCrO 2 + Co 2 CrO 4 phases. On the other hand, the X-ray diffraction patterns of the Mn-substituted samples showed a single P2 type γ-Na x Co 2 O 4 phase. The Fe- and Ni-substituted samples were mostly composed of the Na x Co 2 O 4 phase with small amounts of Co 3 O 4 and CoNiO 2 phases. In the case of the Zn-substituted samples, only the Na x (Co 0.95 Zn 0.05 ) 2 O 4 was mostly composed of the Na x Co 2 O 4 phase with a small amount of the NaO phase. The effects of partial substitution of these 3d transition metals for Co except for Cr in the layered Na x Co 2 O 4 on thermoelectric properties were investigated. The electrical resistivity of these partially substituted samples significantly increased over the entire temperature range, as compared to the non-substituted sample, indicating reduction in carrier concentration. Except for Na x (Co 1-y Fe y ) 2 O 4 , the Seebeck coefficient was enhanced by the partial substitution, and in the case of Na x (Co 1-y Fe y ) 2 O 4 , it is suggested that the decrease in the Seebeck coefficient is associated with reduction in the electron-electron correlation due to the substitution. For the Mn-substituted samples, a small grain size and many pores compared to the non-substituted sample were effective for reducing the thermal conductivity. The figure-of-merit Z was enhanced in the case of the Na x (Co 0.95 Zn 0.05 ) 2 O 4 above 550 K and the maximum dimensionless figure-of-merit ZT, 0.68, was obtained at 965 K.

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.

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 β-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 β-galactosidase. The result of this study showed that BugBuster Master Mix could be a better enzyme extraction reagent for umu test.

To evaluate the protective effects of baicalein and wogonin against benzo[ a ]pyrene- and aflatoxin (AF) B 1 -induced toxicities, the effects of these flavonoids on the genotoxicities and oxidation of benzo[ a ]pyrene and AFB 1 were studied in C57BL/6J mice. Baicalein and wogonin reduced benzo[ a ]pyrene and AFB 1 genotoxicities as monitored by the umu C gene expression response in Salmonella typhimurium TA1535/pSK1002. Baicalein added in vitro decreased liver microsomal benzo[ a ]pyrene hydroxylation (AHH) activity with an ic 50 of 33.9 ± 1.4 μM at 100 μM benzo[ a ]pyrene. Baicalein also inhibited AFQ 1 and AFB 1 -epoxide formation from AFB 1 (50 μM) oxidation (AFO) with ic 50 values of 22.8 ± 1.4 and 5.3 ± 0.8 μM, respectively. However, the in vitro inhibitory effects of wogonin on AHH and AFO activities in liver microsomes were less than those of baicalein as inhibition by 500 μM wogonin was only about 51–65%. Treatment of mice with liquid diets containing 5 mM baicalein and wogonin resulted in 22 and 49% decreases in hepatic AHH activities, respectively. Baicalein treatment resulted in 39 and 32% decreases in AFQ 1 and AFB 1 -epoxide formation from liver microsomal AFO, respectively. Wogonin treatment resulted in 39 and 47% decreases in AFQ 1 and AFB 1 -epoxide formation, respectively. A 1-week pretreatment with wogonin significantly decreased hepatic DNA adduct formation in mice treated with 200 mg/kg of benzo[ a ]pyrene via gastrogavage. These in vitro and in vivo effects suggested that baicalein and wogonin might have beneficial effects against benzo[ a ]pyrene- and AFB 1 -induced hepatic toxicities and that wogonin had a stronger protective effect in vivo .

The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.

We investigated the existence of carcinogenic heterocyclic amines in water samples from a river near Kyoto, where several sewage-treatment plants discharge their effluents and water purification plants downstream use this polluted water as their source. Trp-P-2 is one of the carcinogenic heterocyclic amines included in charred meat and is often excreted in human waste in genotoxic fractions. We analysed Trp-P-2 by reverse-phase HPLC using an ODS column and the UV spectrum. Genotoxic potent activities for blue-rayon extracts of river water were detected in the samples at sampling locations. We identified Trp-P-2 in a genotoxic fraction by MS analysis. The concentrations of Trp-P-2 were calculated in the order of 10−6 mg l−1. These results indicated the existence of carcinogenic heterocyclic amines in the river water. The authors concluded that these carcinogens derive from the effluent of sewage treatment plants including human waste.

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)-catalyzed N-hydroxylation and subsequent esterification by O-acetyltransferase (O-AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH-P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113-120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW"/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O-AT gene in the pOA101-expressing umuC"lacZ gene) in S. typhimurium TA1535. In addition, as an O-AT-deficient strain, we developed the OY1003/1A2 strain by introducing pCW"/1A2:hNPR and pOA101 into O-AT-deficient S. typhimurium TA1535/1,8-DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and > or = 0 nmol isoniazid/min/mg protein as the O-AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2-amino-1,4-dimethylimidazo[4,5-f]quinol ine(MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a::3,2'-d]i midazole,3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole, and 3-amino-1-methyl-5H-pyrido[4, 3-a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O-acetyltransferase.

In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 μM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 μM decreased AHH activity by 50 % to 60 %. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 μM. Naringenin at 100 μM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535 pSK1002 . In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as β-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg ml naringin for 7 days caused 38 % and 49 % decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5, contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38 %. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.

In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 (P450 or CYP) 1A2 and NADPH-P450 reductase and the other (pOA101) a umuC"lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.

Human NAT1 and NAT2 genes were subcloned into pACYC184 vector and the plasmids thus obtained were introduced into Salmonella typhimurium O-acetyltransferase-deficient strain NM6000 (TA1538/1, 8-DNP/pSK1002), establishing new strains NM6001 and NM6002, respectively. We compared the sensitivities of these two strains with those of NM6000 towards carcinogenic nitroarenes and aromatic amines in the SOS/umu response. The induction of umuC gene expression by these chemicals in the presence and absence of the S9 fraction was assayed by measuring the cellular beta-galactosidase activity expressed by the umuC"lacZ fusion gene in the tester strains. 2-Nitrofluorene and 2-aminofluorene induced umuC gene expression more strongly in the NM6001 strain than in the NM6002 strain. In contrast, induction of umuC gene expression by 1, 8-dinitropyrene, 6-aminochrysene and 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline was weaker in the NM6001 strain than in the NM6002 strain. 1-Nitropyrene, 2-amino-6-methyl-dipyrido[1,2-a:3', 2'-d]imidazole, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methyl-9H-pyrido[2,3-b]indole were found to induce umuC gene expression at similar extents in both strains. These results suggest that the newly developed strains can be employed for the studies on mechanisms of genotoxicity of a variety of nitroarenes and aromatic amines, along with the assessment of cancer risk to humans.

It has been identified that the origin of the heterocyclic amines, a group of suspected human carcinogens is high-temperature heating of proteinaceous materials, e. g., food and plants (tabacco-leaves and woods). The heterocyclic amines have been found in human excretions and rain water. We extracted two of these heterocyclic amines, 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2) and 3-amino-1, 4-dimethyl-5H-pyrido[4, 3-b]indole (Trp-P-1) in the water of a sewage teatment plant in Japan by HPLC analysis. The concentration of Trp-P-2 and-1 were estimated at about 10-2μg/L, each. The presence of these compounds shows the possible widespread pollution of surface waters with this class of carcinogens.

Road runoff in rainy days is assumed to include sediment having organic compounds originated from mobile exhaust. We evaluated genotoxicity and ecotoxicity of these compounds by using bacterial assay umu-test, growth inhibition test with algae and acute toxicity test with Thamnocephalus.The effluent from a sewage treatment plant receiving runoff water and runoff from a road in the rainy days were collected. The elute of their particulate and dissolved portions were found to have geno- and ecotoxicity. These toxicities were also found in dust in a collector of tunnel and on the road sediment.

Recently, perfluorooctanoate (PFOA) has been ubiquitously detected in the environment as well as in human serum. Fluorotelomer alcohols (FTOHs), a precursor of PFOA, undergo biodegradation via several metabolic routes which leads to formation of various biodegradation products. The degradation of FTOHs produces an α,β-unsaturated aldehyde that seems possibly to be electrophilic and may react with cellular macromolecules including DNA.We investigated the genotoxicity of three FTOHs (6∶2 FTOH, 8∶2 FTOH and 10∶2 FTOH), PFOA and perfluorooctane sulfonate (PFOS) using theumu test.The FTOHs, PFOA and PFOS showed no significant increases in β-galactosidase activity at 0-1000 μM in the absence of S9 mix. The results were unchanged by the metabolic activation with S9 mix.The genotoxicities of FTOHs, PFOA or PFOS are not detectable using the present method, suggesting that they are unlikely mutagens.

According to the appointment of Ashby et al., 60% of the environmental carcinogens are negative in Ames test. About non-mutagenic carcinogens, it is reported that there is the relationship between carcinogenicity and the ability to produce active oxygen.We choosed organochlorine compounds which are non-mutagenic carcinogens and nitroarenes as test chemicals.As a means to evaluate the ability of chemicals to produce O2-, new strain which can detect soxS induced by O2- is used. And for positive compounds, umu test is tried. And whether the test with the new strain can become a way of screeing of non-mutagenic carcinogens is discussed.As a result of these tests, it is cleared that there are compounds which produce O2- and H2O2 induced by the O2- causes DNA damage. This result suggests that new strain can become a means to screen non-mutagenic carcinogens. Intensity of mutagenic activity of organochlorine compounds in umu test decreased when S9mix was added. When 1-naphthol and quinoline are nitrated, they become to produce O2-. Aromatic compounds existing in tar can get ability to produce O2- after nitrated and can cause DNA damage. Therefore the evaluation of toxic effects of active oxygen should be added to established tests.

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a non-caloric sugar substitute in Japan. The genetic toxicities of stevioside and its aglycone, steviol, were examined with seven mutagenicity tests using bacteria (reverse mutation assay, forward mutation assay, umu test and rec assay), cultured mammalian cells (chromosomal aberration test and gene mutation assay) and mice (micronucleus test). Stevioside was not mutagenic in any of the assays examined. The aglycone, steviol, however, produced dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast cell line (CHL) and the gene mutation assay using CHL. Metabolic activation systems containing 9000 g supernatant fraction (S9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital plus 5,6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis. Steviol was weakly positive in the umu test using S.typhimurium TA1535/pSK1002 either with or without the metabolic activation system. Steviol, even in the presence of the S9 activation system, was negative in other assays, i.e. the reverse mutation assays using S.typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis. Steviol was negative in the mouse micronucleus test. The genotoxic risk of steviol to humans is discussed.

The antigenotoxic effects of curcumin, including the inhibition of SOS induction and mutagenesis by UV light, were investigated in Salmonella typhimurium TA1535/pSK1002 and Escherichia coli K-12 strains. Induction of the SOS gene (umuC) expression was assayed by measuring accumulated beta-galactosidase activity. We found that curcumin blocked umuC induction promoted by UV irradiation in a dose-dependent manner. Also, with another SOS response, Weigle reactivation, we observed that curcumin effectively inhibited phage reactivation by UV irradiation. Furthermore, we tested the effect of curcumin on UV mutagenesis. We showed that mutagenesis induced by UV irradiation was suppressed by the addition of curcumin. Together these results indicate that curcumin acts as an inhibitor of SOS functions including UV mutagenesis.

A highly sensitive umu test system for the detection of carcinogenic/mutagenic aromatic amines has been developed utilizing a new tester strain, Salmonella typhimurium NM2009, possessing an elevated O-acetyltransferase (O-AT) level. NM2009 was constructed by subcloning the bacterial O-AT gene into a plasmid vector pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring an umuC'-'lacZ fusion gene. The system is based on the ability of DNA-damaging agents (genotoxins) to induce umuC gene expression and monitored by measuring the cellular beta-galactosidase activity evoked by the fusion gene. Twenty-two aromatic amine compounds including arylamines, aminoazo dyes, and heterocyclic aromatic amines were tested for inducibility of DNA damage after metabolic activation by rat liver S9 in strain NM2009 and the sensitivity was compared with those of the parent strain TA1535/pSK1002 and the O-AT-defective strain NM2000. NM2009 had about 400 times higher O-AT activity than the parent strain. It was found that NM2009 was much more sensitive to aromatic amines than other strains to induce umuC gene expression after metabolic activation; the chemicals which were extremely sensitive in strain NM2009 include 2-aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene, benzidine, 6-aminochrysene, 2,4-diaminotoluene, 2,6-diaminotoluene, 1-naphthylamine, o-tolidine, 3-MeO-AAB, o-aminoazotoluene, Glu-P-1, Trp-P-1, MeA alpha C, A alpha C, MeIQ, MeIQx, and IQ. In contrast, Trp-P-2 and PhIP showed almost similar sensitivities in three tester strains used in this study. These results suggest that strain NM2009 with high O-acetyltransferase activity is very useful to detect the genotoxic activities of potential mutagenic aromatic amine compounds, which require metabolic activation via the cytochrome P-450/acetyltransferase system.

ガス状癌・変異原性物質に関する研究の一環として, ガス曝露 (バブリング) 法によるタバコ主流煙及び副流煙中のガス状成分の変異原性簡易測定法を検討した。この検討には, Salmonella typhimurium OY413及びTA1535/pSK1002によるumu試験を用いた。また, umu試験から得られた酵素活性の試験菌株の生存率による補正についても過酸化水素を用いて検討を加えた。その結果, 過酸化水素及びタバコ煙中のガス状成分の変異原性測定には通常のumu試験で用いられる600nmの吸光度測定による酵素活性補正よりも平板塗布法による補正の方が正確な変異原性を求め得ることを認めた。インピンジャー6本を直列に接続することによりタバコ煙中のガス状成分の変異原性測定が可能となることも認められた。また, 主流煙及び副流煙共その中に含まれる粒子状成分の方がガス状成分よりも高い変異原性を与えることも認められた。

The genotoxic effects of γ-irradiation were examined using the umu-test, a bacterial short-term test for the detection of genotoxic agents. The principle of the umu-test is based on the induction of the SOS function against DNA damage in Salmonella typhimurium (TA1535/pSK1002 and TA4107/ pSK1002) ; the test measures colorimetrically the expression of umu gene involved directly in SOS mutagenesis. The strains, irradiated aerobically in phosphate buffer, showed a phasic response to γ-irradiation during their growth cycles. The SOS response was the highest level in the lag phase, suggesting that the irradiated lag-phase cells are very sensitive to γ-irradiation and survive via mutation.The hydroxyl radical scavengers, dimethylsulfoxide (DMSO) and ethanol, decreased the SOS response in a dose-related manner. DMSO (0.5 M) added into the aerobic cell suspension decreased the SOS response by about 50% in TA1535/pSK1002 and by about 40% in TA4107/pSK1002 ; the similar proportion was shown in the anaerobic cell suspension containing the scavenger. In addition, the SOS response induced under the aerobic condition was reduced by about 60% in the anaerobic cell suspension containing 0.5 M DMSO. The observations suggest that the SOS response is induced similarly by the direct excitation and the indirect effect by hydroxyl radicals generated during the γ-irradiation of aqueous solution.These results obtained using umu-test were consistent with the phasic response and the irradiation effect reported previously using the survival and DNA strand breaks of bacteria.

In order to address the hypothesis that 6-aminochrysene (6-AC) is converted to genotoxic products by cytochrome P450 enzymes via two activation pathways (N-hydroxylation and epoxidation), the activation of 6-AC and trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene (6-AC-diol) to genotoxic metabolites was examined in rat and human liver microsomal cytochrome P450 enzymes using Salmonella typhimurium TA1535/pSK1002 and TA1535/pSK1002/pNM12 (NM2009) as tester strains. The latter bacteria, an O-acetyltransferase-overexpressing strain, was highly sensitive to metabolites derived from activation of 6-AC, but not those from 6-AC-diol, using liver microsomes from phenobarbital-treated rats or a reconstituted monooxygenase system containing P4502B1 or -2B2, thus suggesting the roles of P450 and acetyltransferase systems in the activation process. 6-AC-diol, on the other hand, was activated very efficiently by liver microsomes prepared from beta-naphthoflavone-treated rats or a reconstituted system containing P4501A1 or -1A2; the activation reaction is considered to proceed through diol-epoxide formation. The contribution of rat P4501A enzymes towards activation of 6-AC-diol was confirmed by the inhibitory effects on the activation process of alpha-naphthoflavone, a specific inhibitor of P4501A-related activities, and antibodies raised against purified P4501A1 and -1A2. In humans, P4501A2 was found to be the major enzyme involved in the activation of 6-AC-diol to genotoxic metabolites while the parent compound 6-AC was activated mainly by P4503A4. Experiments using recombinant P450 proteins expressed in human lymphoblastoid cell lines showed that human P4501A1 could also activate 6-AC-diol to reactive metabolites at almost the same rate measured with P4501A2. In addition, P4502B6 was found to efficiently catalyze the activation of 6-AC to genotoxic metabolites, and P4503A4 was active in the activation of 6-AC-diol as well as 6-AC. Addition of purified rat epoxide hydrolase to the incubation mixture containing purified rat P4501A1 or microsomes expressing human P4501A1 caused inhibition of activation of 6-AC-diol. These results suggest the existence of different enzymatic activation pathways for 6-AC and 6-AC-diol. The former carcinogen may be N-hydroxylated principally by P4502B enzymes in rats and P4503A4 and -2B6 in humans and activation to its ultimate metabolites may proceed through esterification of the N-hydroxy metabolites by an N-acetyltransferase. The 6-AC-diol is metabolized to its ultimate diolepoxide product by P4501A enzymes in rat and human liver microsomes. P4503A4 (humans) and P4503A2 (rats) may also contribute to some extent in the activation of 6-AC-diol, albeit at lower rates than those of P4501A enzymes.

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.

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 β-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.