Most mutagens/carcinogens are metabolically activated by enzymes such as cytochrome P450 to exert their mutagenicity or carcinogenicity. Since the catalytic properties of cytochrome P450 vary among animal species, it was necessary to establish a mutation-detection system carrying human cytochrome P450 to estimate if the chemicals are mutagenic in humans. For this purpose, we introduced genes for human cytochrome P450 together with NADPH-cytochrome P450 reductase to establish humanized Salmonella typhimurium. Using the genetically engineered Salmonella typhimurium, we found that CYP2A6 was involved in the metabolic activation of a variety of nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) contained in tobacco smoke. Analyzing the CYP2A6 gene of subjects who showed a poor metabolic phenotype towards (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazoline-4-one hydrochloride (SM-12502), we discovered a novel mutant allele (CYP2A6*4C) lacking the whole CYP2A6 gene. Taking these results into account, we hypothesized that the subjects carrying the CYP2A6*4C allele had lower risk to tobacco-related lung cancer. In accordance with our hypothesis, our large scale epidemiological studies clearly indicated that smokers homozygous for the CYP2A6*4C allele showed much smaller odds ratios towards cancer risk. Other mutant alleles reducing the CYP2A6 activity besides the CYP2A6*4C allele also reduced the risk for lung cancer in smokers, particularly squamous cell carcinoma and small cell carcinoma frequently seen in smokers. 8-Methoxypsoralen, an inhibitor of CYP2A6, efficiently prevented the occurrence of adenoma caused by NNK in A/J mice.

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Nobiletin, a major component of citrus polymethoxyflavones, possesses anticancer, antiviral and anti-inflammatory activities. To evaluate the chemopreventive potential against lung cancer induced by cigarette smoke, we examined suppressive effects of nobiletin against genotoxicity induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. Male and female gpt delta mice were fed nobiletin at a dose of 100 or 500 ppm in diet for seven days and treated with NNK at a dose of 2 mg/mouse/day, i.p. for four consecutive days. Dietary administration of nobiletin continued at the doses during the NNK treatments and in the following period before sacrifice at day 38. NNK treatments enhanced the gpt mutant frequency (MF) in the lung 19- and 9-fold, respectively, over the values of untreated female and male mice. Interestingly, nobiletin reduced the NNK-induced MFs by 25-45% in both sexes and the reduction at a dose of 100 ppm in females and 500 ppm in males was statistically significant (P<0.05). To further characterize the suppressive effects, we conducted bacterial mutation assay with Salmonella typhimurium YG7108 to examine whether nobiletin inhibits S9-mediated genotoxicity of NNK. Nobiletin as well as 8-methoxypsoralen, an inhibitor of CYP2A, reduced the genotoxicity of NNK by more than 50%. These results suggest that nobiletin may be chemopreventive against NNK-induced lung cancer and also that the chemopreventive efficacy may be due to inhibition of certain CYP enzymes involved in the metabolic activation of NNK.

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The mutagenicity of oxidized adenine, 2-hydroxyadenine (2-OH-Ade), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. 2-OH-Ade was mutagenic with an induced mutation frequency of 0.03-0.8%. 2-OH-Ade mainly caused deletion and A→G mutations. These results indicate that 2-OH-Ade is mutagenic under in vitro replication conditions. The mutation spectrum of 2-hydroxy-dATP in in vitro replication, obtained in a previous study, is discussed.

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We investigated the clastogenic potential of antimycin A, which is well known as a mitochondrial inhibitor (Complex III blocker), utilizing two cultured cell lines, CHL/IU and CHO-WBL. Antimycin A induced chromosome structural aberrations in CHL cells in the presence of S9 mix. However, no chromosome structural aberrations were induced under three different treatment conditions in CHO cells with or without S9 mix. Instead, it induced polyploidy, including endoreduplicated cells, in CHO cells with or without S9 mix. By conducting the comet assay, we found that the antimycin A has no potential to induce DNA damage in these cells. Thus, the chromosome aberrations induced in these cells might be caused by non-DNA-reactive mechanism. Based on these findings, it is suggested that some of the differences between the congenital characteristics of these cells play an important role in the differences of the clastogenic response.

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We recently reported the photomutagenic property of thiabendazole [2-(4-thiazolyl)-1H-benzimidazole, TBZ], a post harvest fungicide, in bacterial and mammalian cells. In the present study we investigated its photomutagenic potential along with several of its structurally-related pesticides including benomyl, mefenacet, tricyclazole, and imazalil. None of the compounds except TBZ showed photomutagenicity with UVA-irradiation in S. typhimurium TA100, TA98, or E. coli WP2uvrA/pKM101. Since TBZ consists of thiazole and benzimidazole rings, we then investigated photomutagenic potential of thiazole, benzimidazole, and 2-phenylbenzimidazole. Only 2-phenylbenzimidazole showed very weak photomutagenicity with UVA-irradiation. These results indicate both benzimidazole ring and thiazole ring are necessary for UVA-activation of TBZ. We measured 8-hydroxydeoxyguanosine (8-OHdG) in salmon sperm DNA exposed to TBZ and UVA by HPLC-ECD, and found that 8-OHdG was not responsible for the photomutagenesis of TBZ.

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In order to assess the potential hazards to human health and aquatic ecosystem, we examined the mutagenic activity of sewage effluents, river waters, sediments and drinking water collected from the Yodo River system, Japan. We also compared the levels of mutagenic activity with the levels of 2-phenylbenzotriazole (PBTA)-type mutagens formed from corresponding dinitrophenylazo dyes via reduction and subsequent chlorination. We assessed mutagenicity in the O-acetyltransferase-overexpressing frameshift strain YG1024 of Salmonella with S9 mix. Sixty-six samples among 133 adsorbates (50%) obtained by the blue rayon hanging method collected from 1996 to 2005 were classified as extreme mutagenicity with more than 100,000 revertants per g blue rayon equivalent (BRE). The average mutagenicity of both sewage effluents and river waters at sites located below sewage plants was 382,400 revertants per g BRE (n=86), which was 4.4 times as higher than the downstream river waters (87,900 revertants per g BRE, n=47). PBTA-1 was detected in 33 samples among 76 (43%), and PBTA-2 was detected in 66 samples among 76 (87%), however, the concentration of these compounds fluctuated widely among the samples. Average concentrations of PBTA-1 and PBTA-2 were also much higher in sewage effluents and river waters at sites located below sewage plants (n=50, PBTA-1, 24.1 ng/g BRE; PBTA-2, 88.4 ng/g BRE) than they were in downstream river water samples (n=26, PBTA-1; 1.3 ng/g BRE, PBTA-2; 19.7 ng/g BRE). PBTA-1 and PBTA-2 accounted for 6% and 26% on average, respectively, of the total mutagenicity in all samples analyzed. Based on the concentrations of the PBTA-type mutagens and the effluent volume discharged from three sewage plants, we estimated that ~5 kg/year of PBTA- type mutagens including PBTA-1, PBTA-2, PBTA-3, PBTA- 4, PBTA-6, PBTA-7 and PBTA-8, were discharged from three sewage plants into rivers. Further studies showed that these PBTA-type mutagens in river water might not easily accumulate in the river sediment and these PBTA-type mutagens were not detected in drinking water. In the final study, we monitored quantitatively the mutagenic potency of water samples collected at twelve sites, including six sites mentioned above, from the Yodo River system using Sep-Pak C18 cartridge columns and Blue-Chitin columns. The average mutagenic activities recovered by these columns were 10,000 and 5,800 revertants/L. These findings demonstrate that the Yodo River system has been continually and heavily polluted with not only polycyclic planar mutagens, but also by a wide range of chemical mutagens released from sewage plants located along the tributaries for many years.

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8-Hydroxydeoxyguanosine (8-OH-dG) is a DNA base modification induced by reactive oxygen species (ROS), and is analyzed in cellular DNA and urine as a marker of oxidative stress. We now report that 8-OH-dG, the ribonucleoside 8-OH-Guo and the free base 8-OH-Gua were detected in fish food products, such as salted dried sardines (Maruboshi) and dried small fishes (Iriko and Shirasu), which are often consumed in Japan. Water extracts of these fish products were analyzed by an HPLC-ECD method, using anion exchange- and reverse phase-columns. Various amounts of 8-OH-Gua (25 ng-22 μg/g), 8-OH-Guo (28-406 ng/g) and 8-OH-dG (5-60 ng/g) were detected in these foods. The amounts of 8-OH-dG and 8-OH-Guo increased upon heating or broiling of these fish products. This is the first report on the presence of genotoxic nucleoside analogues in food. These results provide the warnings that these nucleosides analogues might be food carcinogens, because they are incorporated into DNA by a salvage pathway and show genotoxicity.

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Epidemiological studies suggest that exposure to environmental and occupational electromagnetic fields (EMFs) contributes to the induction of brain tumors, leukemia, and other neoplasms. The aim of this study was to investigate the genotoxic effects of co-exposure to mitomycin C (MMC), a mutagen, and 50-Herz (Hz) EMFs, using an in vivo-ex vivo newborn rat astrocyte micronucleus assay. Three-day-old male Sprague-Dawley rats were co-exposed to 50-Hz EMFs and either 2.0 or 4.0 mg/kg of MMC. Brain cells were dissociated into single cells, cultured for 96 h, and stained with acridine orange and an antibody against glial fibrillary acidic protein. The frequency of micronucleated astrocytes was determined by fluorescence microscopy. The frequency of micronuclei was not increased in rat astrocytes exposed to EMFs alone. However, the frequencies of micronuclei significantly decreased in the rats exposed to 2.0 or 4.0 mg/kg MMC and a 7.5 or 10 mili Tesla (mT) EMF, in comparison to those in the rats exposed to 2.0 or 4.0 mg/kg MMC alone (sham-exposure, 0 mT EMF) for 72 h (p<0.01). Since we previously found that the same range of EMFs enhanced the genotoxicity of cisplatin in the same assay system as that used in the present study, it is suggestSed that effects of EMFs on the genotoxicities of chemicals co-exposed to rats are dependent on the chemicals.

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