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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.

Public concern regarding the transport of air pollutants from mainland East Asia to the leeward area by the prevailing westerlies in spring and winter monsoon has been growing in recent years. We collected total suspended particle (TSP) in Beijing, a metropolis of China located windward of Japan, in spring (late February 2011-May 2011) and in winter (November 2012-early February 2013), then analyzed metals, ions, and organic compounds and mutagenicity, and compared the pollution levels with samples collected at two Japanese metropolises (Osaka and Nagoya) during the same periods. The medians of concentration of TSP and other factors in Beijing were much larger than those in the Japanese metropolises. Especially, the concentrations of polycyclic aromatic hydrocarbons (PAHs) were remarkably high in Beijing in winter, and the median of total PAHs concentration in Beijing was 62-63 times larger than that in the Japanese sites. The mutagenicity of TSP from Beijing toward Salmonella typhimurium YG1024, with and without a mammalian metabolic system (S9 mix), was 13-25 times higher than that from the Japanese sites in winter. These results suggest that air pollution levels in Beijing are very high compared with those at the two Japanese metropolises we evaluated. The diagnostic ratios of PAHs and nitrated polycyclic aromatic hydrocarbons (NPAHs) suggest that the major sources of PAHs and NPAHs in Beijing are different from those at the two Japanese sites in winter, and that the major source in Beijing is coal/biomass combustion.

We recently demonstrated that a novel heterocyclic amine, 5-amino-6-hydroxy-8H-benzo[6,7]azepino[5,4,3-de]quinolin-7-one (ABAQ), is produced from glucose and l-tryptophan by the Maillard reaction at physiological temperature and pH, and that ABAQ was strongly mutagenic for Salmonella strains in the presence of S9 mix. Here, we present the results of three in vivo genotoxicity assays of ABAQ. The comet assay revealed that DNA damage was significantly increased in the livers, kidneys, lungs, and bone marrows of ICR mice, 3h after i.p. injection of ABAQ (50mg/kg body weight (bw)). To evaluate clastogenicity, the peripheral blood micronucleus test was performed, also in ICR mice. ABAQ induced micronucleated reticulocytes (MNRETs) in a dose-dependent manner; the frequency of MNRETs was significantly elevated at all i.p. doses (12.5, 25, and 50mg/kg bw) after 48h. To investigate the mutagenicity of ABAQ in vivo, gpt delta transgenic mice were treated with five consecutive administrations of ABAQ by gavage at doses of 25 or 50mg/kg per week for 3 weeks. The frequencies of gpt mutations (MF) in the liver of mice increased significantly compared with controls, in a dose-dependent manner. No significant increase of gpt MF was detected in the kidneys. Base substitutions predominated; both G:C→A:T and A:T→C:G mutations were significantly increased by ABAQ. The Spi(-) MF was also significantly increased in the liver after ABAQ treatment. If formed in vivo, ABAQ may give rise to adverse genotoxic effects., (Copyright © 2013 Elsevier B.V. All rights reserved.)

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP) was identified as a new potent mutagen toward Salmonella strains in surface soil and airborne particles. Because data of in vivo examination of the genotoxicity of 3,6-DNBeP are limited, micronucleus test was performed in peripheral blood and bone marrow, and comet assay in the lungs of mice treated with 3,6-DNBeP. In male ICR mice intraperitoneally (i.p.) injected with 3,6-DNBeP, the frequency of micronuclated polychromatic erythrocytes (MNPCEs) was increased in the peripheral blood and bone marrow after 24 h in a dose-dependent manner. Compared to controls, the highest dose of 3,6-DNBeP (40 mg/kg B.W.) induced 7.3- and 8.7-fold increases of MNPCE frequency in the peripheral blood and bone marrow, respectively. Furthermore, when 3,6-DNBeP was intratracheally (i.t.) instilled to male ICR mice, 3,6-DNBeP at the highest dose of 0.1 mg/kg body exhibited 3.1-fold increase of DNA tail moment in the lungs at 3 h after the instillation compared to controls. The values of DNA tail moment at 9 and 24 h after the instillation were increased up to 3.5 and 4.2-fold, respectively. These data indicate that 3,6-DNBeP is genotoxic to mammalians in in vivo and suggest that 3,6-DNBeP may be a carcinogenic compound present in the human environment., (Copyright © 2011 Wiley Periodicals, Inc., a Wiley company.)

To clarify the formation of mutagens in the Maillard reaction of glucose and amino acids, 20 amino acids were separately incubated with glucose in the presence or absence of hydroxyl radicals produced by the Fenton reaction. After 1 week at 37 degrees C and pH 7.4, the reaction mixtures of glucose and tryptophan with and without the Fenton reagent showed mutagenicity toward Salmonella typhimurium YG1024 in the presence of a mammalian metabolic system (S9 mix). To identify mutagens in the reaction mixture, blue rayon-adsorbed material from a mixture of glucose, tryptophan, and the Fenton reagent was separated by column chromatography using various solid and mobile phases, and one mutagen, which accounted for 18% of the total mutagenicity of the reaction mixture, was isolated. The chemical structure of the mutagen was determined to be 5-amino-6-hydroxy-8H-benzo[6,7]azepino[5,4,3-de]quinolin-7-one (ABAQ) on the basis of ESI mass, high-resolution APCI mass, (1)H NMR, (13)C NMR, and IR spectral analyses and chemical synthesis of the mutagen. The novel aromatic amine showed high mutagenicity toward S. typhimurium TA98 and YG1024 with S9 mix, inducing 857 revertants of TA98 and 6007 revertants of YG1024/microg, respectively. The mutagenicity of ABAQ was comparable to that of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, which is a mutagenic and carcinogenic hetrocyclic amine in cooked meat and fish formed through the Maillard reaction at high temperature.

The endogenous mutagenic/carcinogenic 9- (4'-aminophenyl) -9H- pyrido [3,4-b] indole (aminophenylnorharman, APNH) is formed from norharman and aniline in the presence of cytochrome P-450s. The major APNH-DNA adduct has been reported to be 2'-deoxyguanosin-8-yl-aminophenylnorhaman (dG-C8-APNH). In addition, demonstrated formation of APNH-RNA adduct and conducted a structural analysis using various spectrometric approaches. The compound produced from guanosine (Guo) and N-acetoxy-APNH, an ultimate mutagenic form of APNH, was concluded to be guanosin-8-yl-APNH (Guo-C8-APNH) on the basis of various spectroscopic analysis. The same adduct was found in the livers of rats administered APNH. The total adduct levels of APNH-RNA were six times higher than total APNH-DNA adducts in the same rat liver samples.

We examined the in vivo mutagenicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and benzo[a]pyrene (BaP) by using transgenic (Tg) zebrafish carrying the mutational target gene rpsL. PBTA-6 is one of the PBTA-type compounds that were recently identified in highly mutagenic river water in Japan. BaP is a well-known contaminant that is frequently found in polluted water. Both compounds are potent mutagens, as determined by using the Ames test employing S9 mix and Salmonella. Adult rpsL Tg zebrafish were exposed to 0, 7, or 10 mg/L PBTA-6 or 0, 1.5, or 3 mg/L BaP for 96 h in a water bath and the mutations in their gills and hepatopancreata were measured 2-4 weeks later. At 3 weeks after exposure, 3 mg/L BaP significantly increased the rpsL mutant frequency (MF) in the gill and hepatopancreas by 5- and 2.3-fold, respectively, as compared to control fish. Sequence analysis showed that BaP mainly induced G:C to T:A and G:C to C:G transversions, which is consistent with the known mutagenic effects of BaP. In contrast, despite its extremely high mutagenic potency in Salmonella strains, PBTA-6 did not significantly increase the MF in the zebrafish gill or hepatopancreas. Although PBTA-6 is 300 times more mutagenic than BaP in the Ames test [T. Watanabe, H. Nukaya, Y. Terao, Y. Takahashi, A. Tada, T. Takamura, H. Sawanishi, T. Ohe, T. Hirayama, T. Sugimura, K. Wakabayashi, Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan, Mutat. Res. 498 (2001) 107-115], calculation of the mutagenicity per mole of compound indicated that PBTA-6 was 33- and <3.7-fold less mutagenic in the zebrafish gill and hepatopancreas, respectively, than BaP.

3,3'-Dichlorobenzidine (DCB), which has been assigned as a possible carcinogen to humans (Group 2B) by IARC, is produced as a raw material in the manufacture of polymers and dye intermediates. In our previous paper, we identified DCB as an indirect-acting mutagenic constituent in the water concentrates from the Waka River, which flows through an industrial area in Wakayama, Japan. In this study, we have identified a novel mutagen in the water samples from the Waka River. Organic chemicals in the river water were adsorbed to blue rayon at the site where effluents from chemical plants and a sewage plant were discharged into the river. The adsorbate was highly mutagenic in Salmonella YG1024 in the presence and absence of S9 mix, inducing 440,000 and 170,000 revertants/g blue rayon equivalent, respectively. Two mutagenic fractions, which accounted for 18% and 12% of the total mutagenicity of the water concentrate in YG1024 with S9 mix, were separated by HPLC with a reversed-phase column following Sephadex LH20 column chromatography. Both fractions were further separated by HPLC using reversed-phase columns. On the basis of spectral analysis and co-chromatography using an authentic chemical standard, one mutagen in the former fraction was identified as DCB and one mutagen in the latter fraction was deduced to be a novel chemical, a 5-nitro derivative of DCB (5-nitro-DCB; 4,4'-diamino-3,3'-dichloro-5-nitrobiphenyl). 5-Nitro-DCB showed strong mutagenicity in YG1024 especially with S9 mix, inducing 24,200 revertants/microg. 5-Nitro-DCB was detected in water concentrates in the range from less than detection limit to 6.9 microg/g of blue rayon. DCB was also detected in the range from 13.2 to 104 micro/g of blue rayon. These results demonstrate that Waka River water might be continually contaminated with the indirect-acting mutagens DCB and 5-nitro-DCB as major mutagenic constituents of the river water.

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. alpha-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.

To clarify the mutagenic potential of surface soil in residential areas in Kyoto city, surface soil samples were collected twice or three times from 12 sites, and their organic extracts were examined by the Ames/Salmonella assay. Almost all (>92%) samples showed mutagenicity in TA98 without and with S9 mix, and 8/25 (32%) samples showed high (1000-10,000 revertants/g of soil) or extreme (>10,000 revertants/g of soil) activity. Moreover, to identify the major mutagens in surface soil in Kyoto, a soil sample was collected at a site where soil contamination with mutagens was severe and continual. The soil extract, which showed potent mutagenicity in TA98 without S9 mix, was fractionated by diverse column chromatography methods. Five major mutagenic constituents were isolated and identified to be 1,6-dinitropyrene (DNP), 1,8-DNP, 1,3,6-trinitropyrene (TNP), 3,9-dinitrofluoranthene (DNF), and 3,6-dinitrobenzo[e]pyrene (DNBeP) by co-chromatography using high performance liquid chromatography and spectral analysis. Contribution ratios of 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP to total mutagenicity of the soil extract in TA98 without S9 mix were 3, 10, 10, 10, and 6%, respectively. These nitroarenes were detected in surface soil samples collected from four different residential sites in other prefectures, and their contribution ratios to soil mutagenicity were from 0.7 to 22%. These results suggest that surface soil in residential areas in Kyoto was widely contaminated with mutagens and there were some sites where surface soils were heavily polluted. 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP may be major mutagenic constituents that contaminate surface soil in Kyoto and other residential areas.

A novel heterocyclic amine, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), which is formed from nonmutagenic 9H-pyrido[3,4-b]indole (norharman) and aniline, is mutagenic to bacteria and mammalian cells and potently carcinogenic in rats. APNH is detected in human urine samples, suggesting that humans are continuously exposed to APNH. In the present study, (32)P-postlabelin analysis revealed that the levels of APNH-DNA adduct 24 hr after the treatment with APNH (1, 5 and 20 mg/kg body weight) in male ICR mice were increased in a dose-dependent manner in the colon and liver. Based on these findings, we determined the tumor-initiating potency of APNH in an inflammation-related and two-stage mouse colon carcinogenesis model. Male Crj: CD-1 (ICR) mice were given a single intragastric administration (1, 2, 5 or 10 mg/kg body weight) of APNH and subsequent 1-week oral exposure to dextran sodium sulfate (DSS, 2% in drinking water). Treatment with APNH and DSS resulted in numerous colon tumor development: the incidence and multiplicity of the tumors were the highest in the mice received 10 mg/kg body weight of APNH and followed by DSS. Development of colon tumors was dose-dependent of APNH. Seven of 9 (77.8%) colonic adenocarcinomas developed in mice treated with APNH (10 mg/kg body weight) and DSS had beta-catenin gene mutations at codons 32 and 37, being predominantly transversion. These findings indicate that APNH has an initiating activity in inflamed mouse colon and the APNH-DNA adduct formation correlates with its tumorigenic potential., ((c) 2007 Wiley-Liss, Inc.)

Mutagenic/carcinogenic 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnorharman (APNH)] is formed from norharman and aniline in the presence of cytochrome P450 3A4/1A2. Because both precursors are widely distributed in the environment, human exposure is unavoidable. To clarify APNH formation in the human body, amounts of the compound in 24-h human urine collected from smokers and nonsmokers, eating a normal diet, were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry. In addition, norharman and aniline were also analyzed by high-performance liquid chromatography and gas chromatography, respectively. APNH could be detected in all urine samples at levels 49 to 449 pg for smokers and 21 to 594 pg for nonsmokers per 24-h urine, respectively. The amounts of norharman and aniline were 46 to 185 ng and 0.70 to 8.10 microg for smokers and 52 to 447 ng and 0.49 to 5.72 microg for nonsmokers, respectively, per 24-h urine (none of the levels differing significantly between smokers and nonsmokers). To exclude exogenous exposure to norharman and aniline, we analyzed the levels of APNH, norharman, and aniline in urine samples collected from inpatients receiving parenteral alimentation. Similar to the healthy volunteers, all urine samples contained 12 to 338 pg of APNH, 6 to 75 ng of norharman, and 0.33 to 1.86 microg of aniline per 24-h urine. These results suggest that APNH should be considered as a novel endogenous mutagen/carcinogen; thus, it is very important to determine the biological significance of this carcinogen for human cancer development.

We previously identified 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA) congeners as major mutagens in water concentrates from several rivers that flow in three different areas, i.e. Kyoto, Aichi, and Fukui Prefectures, in Japan. In synthesis studies, these PBTAs were shown to be formed from corresponding dinitrophenylazo dyes via non-chlorinated derivatives (non-ClPBTAs). However, only non-ClPBTA-1, i.e. 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole, had been detected as a minor contaminant in the Nishitakase River in Kyoto. In this study, analysis of mutagens in water concentrate from the Ho River, which flows through an area with a textile dyeing industry in Shizuoka Prefecture, Japan, allowed the isolation of four compounds (I, II, III, and IV). These four mutagens were identified as 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-2), 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-3), 2-(2-acetylamino-4-amino-5-methoxyphenyl)-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-4), and 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-6-amino-4-bromo-2H-benzotriazole (non-ClPBTA-7) by spectral data and co-chromatography using synthesized standards. Non-ClPBTA-3 and -7 were highly mutagenic in Salmonella typhimurium YG1024, inducing 159,000 and 178,000 revertants/microg, respectively, in the presence of S9 mix. Like PBTAs, non-ClPBTAs might have been produced from azo dyes during industrial processes in dyeing factories and released into rivers.

Male F344 5-week-old rats were X-irradiated, and 16 weeks after the first dose, azoxymethane (AOM) was injected or 2-amino-1 -methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) was given by intragastric intubation. Tumors in the pylorus of the glandular stomach were observed in 4 out of the 29 animals receiving X-rays + AOM and in 4 out of the 25 animals receiving X-rays + PhIP, 12 months after administration. No such lesions were found in the chemical or X-ray alone groups. Intestinal metaplasia and some induced tumors were positive for CDX2. It was concluded that the presence of intestinal metaplasia may increase sensitivity to the induction of gastric tumors by colon carcinogens.

We previously identified 1,3-, 1,6-, and 1,8-dinitropyrene (DNP) isomers as major mutagens in surface soil in three metropolitan areas of Japan. In the present study, an organic extract from surface soil collected at a park in Takatsuki in Osaka Prefecture, which showed extremely high mutagenicity in Salmonella typhimurium TA98 in the absence of mammalian metabolic system (S9 mix), was investigated to identify major mutagens. A new powerful bacterial mutagen, as well as 1,6- and 1,8-DNP isomers, was isolated from the organic extract (1.8 g) of the soil sample (2.2 kg) by column chromatography. On the basis of mass spectra, the new mutagen, which accounted for 15% of the total mutagenicity of the soil extract, was thought to be a dinitrated polycyclic aromatic hydrocarbon with a molecular weight of m/z 342. The mutagen was synthesized from benzo[e]pyrene by nitration and was determined to be 3,6-dinitrobenzo[e]pyrene (DNBeP) based on its 1H NMR spectrum. The mutagenic potency of 3,6-DNBeP in the Ames/Salmonella assay was extremely high, in that it induced 285,000 revertants/nmol in TA98 and 955,000 revertants/nmol in YG1024 without S9 mix and was comparable to those of DNP isomers, which are some the most potent bacterial mutagens reported so far. In addition to the soil sample from Takatsuki, 3,6-DNBeP was also detected in surface soil samples collected at parks in four different cities, i.e., Izumiotsu and Takaishi in Osaka Prefecture and Nagoya and Hekinan in Aichi Prefecture, and accounted for 22-29% of the total mutagenicity of these soil extracts in TA98 without S9 mix. These results suggest that 3,6-DNBeP is a major mutagen in surface soil and may largely contaminate the surface soil in these two regions in Japan.

We describe a method for the simultaneous determination of eight kinds of phenylbenzotriazole-type mutagens (PBTA-1, -2, -3, -4, -5, -6, -7 and -8) in river water based on liquid chromatography-tandem mass spectrometry (LC/MS/MS). The application of dopant-assisted atmospheric pressure photoionization (APPI) for the detection of the PBTAs was studied. The APPI technique provided higher PBTA signal intensities than those obtained with an electrospray ionization (ESI) source, and the APPI method was used for the determination of the PBTAs. A solid-phase extraction procedure was used for the extractions of PBTA-1 through -8 from river water. The procedure was rapid and the relative standard deviations were below 15%. The detection limits of PBTA-1 through -8 in river water using the proposed method were found to range from 0.04 to 0.5 ng L(-1) and PBTAs were successfully detected in river water at sub-ng L(-1) levels.

A review of the literature on the mutagenicity/genotoxicity of surface waters is presented in this article. Subheadings of this article include a description of sample concentration methods, mutagenic/genotoxic bioassay data, and suspected or identified mutagens in surface waters published in the literature since 1990. Much of the published surface water mutagenicity/genotoxicity studies employed the Salmonella/mutagenicity test with strains TA98 and/or TA100 with and/or without metabolic activation. Among all data analyzed, the percentage of positive samples toward TA98 was approximately 15%, both in the absence and the presence of S9 mix. Those positive toward TA100 were 7%, both with and without S9 mix. The percentage classified as highly mutagenic (2500-5000 revertants per liter) or extremely mutagenic (more than 5000 revertants per liter) was approximately 3-5% both towards TA98 and TA100, regardless of the absence or the presence of S9 mix. This analysis demonstrates that some rivers in the world, especially in Europe, Asia and South America, are contaminated with potent direct-acting and indirect-acting frameshift-type and base substitution-type mutagens. These rivers are reported to be contaminated by either partially treated or untreated discharges from chemical industries, petrochemical industries, oil refineries, oil spills, rolling steel mills, untreated domestic sludges and pesticides runoff. Aquatic organisms such as teleosts and bivalves have also been used as sentinels to monitor contamination of surface water with genotoxic chemicals. DNA modifications were analyzed for this purpose. Many studies indicate that the 32P-postlabeling assay, the single cell gel electrophoresis (comet) assay and the micronucleus test are sensitive enough to monitor genotoxic responses of indigenous aquatic organisms to environmental pollution. In order to efficiently assess the presence of mutagens in the water, in addition to the chemical analysis, mutagenicity/genotoxicity assays should be included as additional parameters in water quality monitoring programs. This is because according to this review they proved to be sensitive and reliable tools in the detection of mutagenic activity in aquatic environment. Many attempts to identify the chemicals responsible for the mutagenicity/genotoxicity of surface waters have been reported. Among these reports, researchers identified heavy metals, PAHs, heterocyclic amines, pesticides and so on. By combining the blue cotton hanging method as an adsorbent and the O-acetyltransferase-overproducing strain as a sensitive strain for aminoarenes, Japanese researchers identified two new type of potent frameshift-type mutagens, formed unintentionally, in several surface waters. One group has a 2-phenylbenzotriazole (PBTA) structure, and seven analogues, PBTA-type mutagens, were identified in surface waters collected at sites below textile dyeing factories and municipal wastewater treatment plants treating domestic wastes and effluents. The other one has a polychlorinated biphenyl (PCB) skelton with nitro and amino substitution group and it was revealed to be 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl derived from chemical plants treating polymers and dye intermediates. However, the identification of major putative mutagenic/genotoxic compounds in most surface waters with high mutagenic/genotoxic activity in the world have not been performed. Further efforts on chemical isolation and identification by bioassay-directed chemical analysis should be performed.

A novel mutagenic compound, 9-(4'-aminophenyl)-9H- pyrido[3,4-b]indole (aminophenylnorharman, APNH), is shown to be formed by the in vitro enzymatic reaction of 9H-pyrido[3,4-b]indole (norharman) and aniline. APNH generates DNA adducts (dG-C8-APNH), and is potently genotoxic to bacteria and mammalian cells. APNH has also been demonstrated to be formed in vivo from norharman and aniline, and suggested to be a new type of endogenous mutagenic compound. To determine its carcinogenic activity, long-term administration of APNH was investigated in 93 male and 90 female F344 rats. Rats were fed diets containing 0, 20 or 40 p.p.m. from 7 weeks of age. All animals were killed after 85 weeks treatment and necropsy was performed. Hepatocellular carcinomas (HCCs) were induced at incidences of 10 and 79% in male rats fed 20 and 40 p.p.m. APNH, and 34% in female rats fed 40 p.p.m. of APNH, respectively. In addition, colon adenocarcinomas were found at incidences of 3 and 9% in male rats, and 4 and 13% in female rats fed 20 and 40 p.p.m. of APNH, respectively. Other tumors, including thyroid carcinomas and mononuclear cell leukemia, were also seen in rats fed APNH. Polymerase chain reaction-single strand conformation polymorphism analysis revealed beta-catenin gene mutations in 24% of HCCs and K-ras, beta-catenin and Apc gene mutations were found in 22, 44 and 33% of colon cancers induced by APNH, respectively. Most mutations occurred at G:C base pairs. beta-Catenin protein accumulations in the nucleus and cytoplasm were also revealed in both liver and colon tumors. Thus, APNH induced liver and colon cancers with K-ras, beta-catenin and Apc gene mutations in F344 rats.

Mutagenic 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), formed from norharman and aniline in the presence of S9 mix, is thought to be accountable for the co-mutagenic action of norharman. Our previous studies suggest that cytochrome P-450s (CYPs) are involved in the generation of APNH. In order to identify the responsible CYP species in the present study, norharman (8 mg) and aniline (4 mg) were incubated with individual recombinant human CYPs (2 nmol) at 37 degrees C for 20 min. Formation of APNH was observed with CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2D6, CYP2E1 and CYP3A4, but not with CYP2A6, CYP2C9 and CYP2C19. The amounts of APNH from norharman and aniline were 33 ng for CYP1A1, 15 ng for CYP3A4, 7 ng for CYP2D6, 6 ng for CYP1A2 and 5 ng for CYP2B6. APNH formation in the presence of CYP1B1 and CYP2E1 was very low at around one fiftieth of that with CYP3A4. When CYP selective chemical inhibitors, such as furafylline for CYP1A2 and ketoconazole for CYP3A4, were added to the reaction mixture of norharman, aniline and human microsomes, formation of APNH was decreased to 14 and 16% of the control level, respectively. Moreover, human lung microsomes also showed the activity of APNH formation from norharman and aniline, albeit at only one hundredth of that with liver microsomes. In general, content in human liver microsomes is rather high for CYP3A4 and CYP1A2 but relatively low for CYP2D6 and CYP2B6, at about 30, 10, 1.5% and less than 1% of the total CYP, respectively. Although CYP1A1 showed the highest APNH formation activity, its expression in human liver is reported to be below the level of detection. Based on these observations, it is suggested that the practical major contributors to the formation of APNH from norharman and aniline are CYP3A4 and CYP1A2, the responsible reactions mainly occurring in the liver.

2-[2-(Acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl (ADDB) are two compounds, which show strong mutagenicity toward bacteria, that have been identified as major mutagens in river water in Japan. In the present study, we examined the genotoxicity of PBTA-6 and ADDB in goldfish (Carassius auratus) by the micronucleus test and single-cell gel electrophoresis (comet assay). The frequencies of micronuclei in gill cells gradually increased until 96h after i.p. injection of PBTA-6 and ADDB at doses of 50mg/kg body weight, and then decreased 144h after injection. PBTA-6 induced micronuclei in gill cells dose-dependently at a dose range of 1-100mg/kg body weight, giving significantly high frequencies at doses of 50 and 100mg/kg body weight. On the other hand, no significant increase was observed in the peripheral erythrocytes of goldfish exposed to PBTA-6 or ADDB. In the comet assay, values of DNA tail moment and tail length in peripheral erythrocytes increased significantly until 6h after the i.p. injection of PBTA-6 (50mg/kg body weight), only to decrease by 9h after injection. Both the DNA tail moment and tail length were dose-dependently increased by injections of PBTA-6 at doses ranging from 1 to 50mg/kg. Significantly high values for tail moment and tail length were found in peripheral erythrocytes 3h after an i.p. injection of ADDB and persisted for up to 6h. These results show that both PBTA-6 and ADDB have genotoxic effects in goldfish.

Norharman, widely distributed in our environment such as cigarette smoke and cooked foods, is not mutagenic to Salmonella strains, but becomes mutagenic to Salmonella typhimurium TA98 and YG1024 with S9 mix in the presence of aromatic amines, including aniline and o-toluidine. Therefore, we have designated norharman as a "co-mutagen". Since, humans are simultaneously exposed to norharman and aromatic amines in daily life, it is important to clarify the mechanisms of its co-mutagenic action to further understanding of the potential genotoxic effects in humans. Regarding the mechanisms of this action of norharman with aniline, a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole[aminophenylnorharman (APNH)] is produced by their interaction, and converted to the hydroxyamino derivative which eventually forms the DNA adduct, dG-C8-APNH through possible ultimate reactive forms with esterification, and this induces mutations. Also other aminophenyl-beta-carboline compounds, such as 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole[amino-3'-methylphenylnorharman (3'-AMPNH)], 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole [amino-2'-methylphenylnorharman (2'-AMPNH)], 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole[aminophenylharman (APH)] and 9-(4'-amino-3'-methylphenyl)-1-methyl-9H-pyrido[3,4-b]indole[amino-3'-methylphenylharman (AMPH)], have been found on reaction of norharman or harman with aniline or toluidine isomers. These compounds showed mutagenic and clastogenic actions in bacterial and mammalian cells. Among them, APNH demonstrated the most potent activity, and it was most extensively studied. When APNH was administered as a single dose to F344 rats, severe testicular toxicity was observed after 6 days. Moreover, liver preneoplastic lesions (GST-P-positive foci) in the liver clearly developed in animals fed 10-50 ppm of APNH in the diet for 4 weeks. Since, APNH was detected in 24 h urine of rats upon simultaneous administration with norharman and aniline by gavage, it is likely to be also produced from norharman and aniline in the human body. From these findings, it is suggested that aminophenyl-beta-carboline derivatives may be classified as one of the novel types of endogenous mutagens and carcinogens.

Aminophenylnorharman (APNH) is formed from non-mutagenic norharman and aniline, and is mutagenic to Salmonella typhimurium TA98 with S9 mix. Norharman and aniline are present in cigarette smoke and cooked foods and both compounds are detected in human urine samples, suggesting that APNH could be a mutagenic and carcinogenic human risk factor. The purpose of the present study was to determine the in vivo mutagenicity of APNH. Male gpt delta transgenic mice were fed a diet containing 10 or 20 p.p.m. APNH for 12 weeks. The gpt mutant frequency (MF) in the liver increased 10-fold in 20 p.p.m. APNH-treated mice, which was almost equivalent to the MF observed in the liver of the same transgenic mice treated with 300 p.p.m. 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline for 12 weeks. In the colon mucosa, the gpt MF increased approximately 5-fold in 20 p.p.m. APNH-treated mice. Our results suggest that APNH is a strong hepatic mutagen in mice. The APNH-induced gpt mutations in the liver were dominated by G:C to T:A transversions, followed by G:C to A:T transitions. They also included single G:C deletions in G:C run sequences and 2 bp deletions: GCGC to GC and CGCG to CG. The Spi- deletion MF in the liver was 13-fold higher in 20 p.p.m. APNH-treated mice, relative to the control, and were dominated by single base pair deletions, in particular, in G:C run sequences. Large deletions were rare. The mutational characteristics induced by APNH are compared with those induced by other heterocyclic amines, and the human risk of APNH is discussed.

To clarify the mutagenic potential of surface soil in the Kinki region of Japan, particularly in Osaka and neighboring cities, 62 surface soil samples were collected and their organic extracts were examined by the Ames/Salmonella assay. All of the samples were mutagenic toward TA98 in both the presence and absence of a mammalian metabolic activation system (S9 mix). While all of the samples showed mutagenicity toward TA100 with S9 mix, only 45/62 (73%) were mutagenic without S9 mix. Fifty (81%) of the samples showed higher activity toward TA98 than TA100. The mean values of the mutagenicities of soil samples collected in Osaka prefecture (n=35) toward TA98 with and without S9 mix were 2315 and 1630 revertants per gram of soil, respectively, and these were 2.9 and 2.6 times as high as the values for samples from other prefectures (n=27), respectively. Three dinitropyrene (DNP) isomers, i.e. 1,3-, 1,6- and 1,8-DNP, and 3-nitrobenzanthrone (NBA) in the surface soil samples were quantified by fluorometric detection of the corresponding amino compounds, i.e. diaminopyrene isomers and 3-aminobenzanthrone, using high-performance liquid chromatography (HPLC). The three DNP isomers were detected in all of the soil samples (n=26) that were mainly collected in Osaka prefecture, and the amounts of 1,3-, 1,6- and 1,8-DNP were 6-1526, 11-1772 and 10-2092pg/g of soil, respectively. The contribution ratios of 1,3-, 1,6- and 1,8-DNP to the mutagenicity of soil extracts toward TA98 without S9 mix were 0.2-12, 0.3-12 and 0.5-27%, respectively. The amount of 3-NBA in soil samples (n=8) was 144-1158pg/g of soil, and the contribution ratio of 3-NBA to the mutagenicity of soil extracts was 2-38%. These results suggest that the surface soils in the Kinki region were highly polluted with mutagens and the pollution levels in Osaka prefecture were higher than those in other areas. DNP isomers and 3-NBA may be major mutagens that contaminate surface soil in this region.

We previously reported on the isolation and structural determination of five 2-phenylbenzotriazole (PBTA)-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4 and PBTA-6) in blue rayon/cotton adsorbed substances collected from surface waters at sites located downstream of sewage treatment plants. We also noted that PBTA-1 and PBTA-2 were discharged from sewage treatment plants and subsequently diluted or decomposed while moving down the Yodo River system. However, it has not been investigated whether they are commonly discharged from sewage treatment plants into rivers. The main purpose of this study was to make a comprehensive survey of levels and behavior of PBTA-type mutagens in effluents discharged from the sewage treatment plant located along the bank of the Uji River, one tributary of the Yodo River system. Water samples were collected at the outlet of the sewage treatment plant for 16 consecutive days in May 1999 and 11 consecutive days in December 1999. Organic constituents were obtained via sorption to blue rayon and subsequent methanol elution. Extract mutagenic activity was measured using Salmonella typhimurium YG1024 with metabolic activation. PBTA-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4, PBTA-5 and PBTA-6) were quantified by HPLC with electrochemical detection, followed by HPLC purification on reverse-phase columns. The study showed that PBTA-2, PBTA-3, PBTA-4 and PBTA-6 were detected in most samples. The total contribution of these four PBTA-type mutagens to overall extract mutagenicity is on average 33% for the May 1999 sample and 58% for the December 1999 sample. The individual PBTA compounds that had the largest contribution to the overall mutagenicity were PBTA-3 and PBTA-4, accounting for 11 and 16% in May 1999, and 25 and 26% in December 1999. A further comparative study was done in December 1999 using the blue rayon hanging method and the results were similar to those obtained using the blue rayon column method. In conclusion, the present study showed that PBTA-2, PBTA-3, PBTA-4 and PBTA-6 were commonly discharged from a sewage treatment plant into the Uji River, and they accounted for a substantial portion of the effluent mutagenicity.

A mutagenic heterocyclic amine (HCA), 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), is produced in the presence of S9 mix by the reaction of norharman and aniline, both of which are nonmutagenic and abundantly present in our environment. It has been previously reported that APNH-DNA adducts were detected in DNA of Salmonella typhimurium strain incubated with APNH and S9 mix. In the present study, we examined the structures of APNH-DNA adducts using the (32)P-postlabeling method and various spectrometry techniques. When the reaction mixture of N-acetoxy-APNH and 2'-deoxyguanosine 3'-monophosphate (3'-dGp) was analyzed, three adduct spots (two major and one minor) were observed by (32)P-postlabeling under modified-standard conditions. No adduct formation was observed for reaction mixtures of N-acetoxy-APNH with 3'-dAp, 3'-dTp, or 3'-dCp. The two major adduct spots (spots 1 and 2) detected by TLC were extracted and subjected to HPLC along with the standards 3',5'-pdGp-C8-APNH and 5'-pdG-C8-APNH, which were independently chemically synthesized. On the basis of the results of co-chromatography, spots 1 and 2 were identified to be 5'-monophosphate and 3',5'-diphosphate forms of dG-C8-APNH. When the extract of spot 2 (3',5'-pdGp-C8-APNH) was further digested with nuclease P1 and phosphodiesterase I, a spot corresponding to spot 1 (5'-pdG-C8-APNH) was newly observed on TLC. From these observations, both of the two major spots were concluded to be dG-C8-APNH. A similar DNA adduct pattern to that apparent in vitro was observed in various organs of F344 rats fed 40 ppm of APNH for 4 weeks. The levels of APNH-DNA adducts were highest in the liver and colon, with RAL values of 1.31 +/- 0.26 and 1.32 +/- 0.11 adducts/10(7)nucleotides, respectively. Thus, APNH was demonstrated to form DNA adducts primarily at the C-8 position of guanine residues in vitro and in vivo, like other mutagenic and carcinogenic HCAs.

To clarify their mutagenic potential, samples of water from the Mawatari, Asuwa and Kitsune rivers, which flow through the central area of Fukui, Japan, were seasonally collected at six sites using blue rayon from July 1998 to August 2000. Forty-five of 52 (87%) of the water samples exhibited mutagenicity toward Salmonella typhimurium YG1024 and YG1029 with and without S9 mix, and the highest potencies were observed in YG1024 with S9 mix. The samples collected in summer and autumn tended to be more mutagenic than those collected in winter and spring. Fractionation using high-performance liquid chromatography (HPLC) suggests that several compounds are responsible for the mutagenicity of river water samples, and some of the major mutagens seem to be common among the samples. Three 2-phenylbenzotriazole (PBTA)-type mutagens, 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-3), 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) and 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6), were quantified in samples collected between July 1998 and April 1999. At least one of these PBTA-type mutagens was detected in 23/24 (96%) of the samples. The amounts of PBTA-3, -4 and -6 were <0.08-58.7, <0.1-15.0 and <0.07-467.9 ng/g of blue rayon, respectively, and high levels of PBTA congeners were detected in the samples collected from each river in July and November 1998. The contributions of these PBTA congeners to the mutagenicity of water samples were also high in July and November 1998. The highest total contribution was observed for samples from the Asuwa river (67.6%). These findings suggest that these three rivers were continually and heavily contaminated with mutagens, and PBTA congeners were some of the major mutagens in these rivers.

Aminophenylnorharman (APNH) is a newly identified mutagenic heterocyclic amine formed by coupling of norharman with aniline in the presence of S9 mix. Furthermore, mutagenic amino-3'-methylphenylnorharman (AMPNH) and aminophenylharman (APH) have been identified from a reaction mixture of norharman and o-toluidine and that of harman and aniline, respectively, with S9 mix. Among these three heterocyclic amines, APNH shows most potent mutagenic activity towards Salmonella typhimurium TA98 and YG1024 with S9 mix. In the present study, the induction of sister chromatid exchanges (SCEs) by APNH was examined in Chinese hamster lung (CHL) cells in vitro, comparing it to those of AMPNH and APH. On incubation with rat S9 for 6h, followed by a recovery culture period of 18h, a dose-dependent effect was found at concentrations between 0.00125 and 0.01 microg/ml for APNH and between 0.3125 and 5 microg/ml for AMPNH and APH. The approximate chemical concentrations leading to a three-fold of control SCE levels calculated from slopes of the linear regressions of induced SCEs were 0.005 for APNH, 0.51 for AMPNH and 1.7 microg/ml for APH. Because of the very strong SCE-causing ability of APNH, we further explored its genotoxicity by examining the induction of chromosome aberrations in CHL cells. A dose-dependent effect was found for chromosome aberrations at concentrations between 0.00125 and 0.04 microg/ml of APNH. The aberrations observed were primarily chromatid exchanges (cte) and breaks (ctb). In conclusion, the potency of SCE induction and clastogenic activity induced by APNH is stronger than Actinomycin D, Mitomycin C (MMC) or 1,8-dinitropyrene which are considered to be the potent clastogens in the literature. Further studies are needed for elucidating mechanisms of the genotoxic actions of these compounds and for evaluating their potential hazards to human health.

Water samples from the Waka River, which runs through an area housing many chemical industry facilities in Wakayama, Japan, have been found to show significant mutagenicity, especially without a mammalian metabolic activation system (S9 mix) in the Salmonella typhimurium YG1024 strain. Mutagens in the river water were adsorbed to 3 kg of blue cotton, extracted with methanol/ammonia, and separated by several low- and high-pressure liquid chromatography steps with reversed-phase columns. One mutagen (0.6 mg), accounting for 50% of the total mutagenicity of the adsorbed materials, was isolated. On the basis of the mass, high-resolution mass, (1)H NMR and (13)C NMR spectra, the chemical was determined to have a polychlorinated biphenyl skeleton with nitro and amino substitution groups. Well-designed chemical synthesis of the putative mutagen revealed it to be 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl. This novel compound exerted strong mutagenicity without the S9 mix, inducing 66,000 and 140,000 revertants/nmol in S. typhimurium TA98 and YG1024, respectively. Moreover, this polychlorinated biphenyl derivative was proven to activate the human aryl hydrocarbon receptor-mediated transcription in a lac Z reporter gene assay with an efficiency almost the same as that of beta-naphthoflavone, well-known to be a synthetic aryl hydrocarbon receptor agonist. It is possible that the mutagen is formed unintentionally via postemission modification of drainage water containing parent chemicals, such as 3,3'-dichlorobenzidine or 3,3'-dichloro-4,4'-dinitrobiphenyl, which are known to be raw materials in the manufacture of polymers and dye intermediates in chemical plants.

Pan-roasted beef showed a lower mutagenicity after various degrees of cooking than charcoaled one. The high mutagenicity of charcoaled beef was due to the formation of more heterocyclic amines, especially AalphaC (2-amino-9H-pyrido- [2,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) because of rapid and direct heating on the surface of the meat at a high temperature. Seasoning decreased mutagenicity of pan-roasted beef except the very well done sample with unchanged heterocyclic amine contents, but increased mutagenicity of charcoaled beef with decreased levels of AalphaC and PhIP, probably due to the change of heterocyclic amine precursors or alternatively to the occurrence of other mutagens.

Norharman (9H-pyrido[3,4-b]indole), which is a heterocyclic amine included in cigarette smoke or cooked foodstuffs, is not mutagenic itself. However, norharman reacts with non-mutagenic aniline to form mutagenic aminophenylnorharman (APNH), of which DNA adducts formation and hepatocarcinogenic potential are pointed out. We investigated whether N-OH-APNH, an N-hydroxy metabolite of APNH, can cause oxidative DNA damage or not, using 32P-labeled DNA fragments. N-OH-APNH caused Cu(II)-mediated DNA damage. When an endogenous reductant, beta-nicotinamide adenine dinucleotide (NADH) was added, the DNA damage was greatly enhanced. Catalase and a Cu(I)-specific chelator inhibited DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). Typical -*OH scavenger did not inhibit DNA damage. These results suggest that the main reactive species are probably copper-hydroperoxo complexes with DNA. We also measured 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation by N-OH-APNH in the presence of Cu(II), using an electrochemical detector coupled to a high-pressure liquid chromatograph. Addition of NADH greatly enhanced 8-oxodG formation. UV-VIS spectra and mass spectra suggested that N-OH-APNH was autoxidized to nitrosophenylnorharman (NO-PNH). We speculated that NO-PNH was reduced by NADH. Cu(II) facilitated the redox cycle. In the presence of NADH and Cu(II), very low concentrations of N-OH-APNH could induce DNA damage via redox reactions. We conclude that oxidative DNA damage, in addition to DNA adduct formation, may play an important role in the expression of genotoxicity of APNH.

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.

We performed the in vitro micronucleus (MN) test on 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)-ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), which are newly identified water pollutants from the Nishitakase river in Kyoto, Japan, and on their possible mother compounds (AZO DYE) and intermediates (non-ClPBTAs). We tested these compounds in the absence and presence of S9 mix in two Chinese hamster cell lines CHL and V79-MZ and scored MN, polynuclear and karyorrhectic (PN), and mitotic (M) cells. PBTA-2 in the absence of S9 mix induced the strongest responses in both cell lines. It was also a strong inducer of binucleate cells in PN cells in both cell lines, which suggested that it induced polyploidy. PBTA-1 showed clear positive results only in the absence of S9 mix and only in V79-MZ cells, inducing aneuploidy. In CHL cells AZO DYE-1 significantly induced MN cells in the presence of S9 mix, and AZO DYE-2 induced MN and PN cells, including binucleate cells and cells with a multilobed nucleus, in the absence of S9 mix. In V79-MZ cells, AZO DYE-1 and -2 induced primarily M cells in the presence of S9 mix. 9% of the M cells treated with 50 microg/ml AZO DYE-1 showed endoreduplication. AZO DYE-2 at 200 microg/ml condensed the chromatin in 100% of the cells. The non-ClPBTAs were a bit more cytotoxic than the other compounds and induced a slight increase in MN cells in both cell lines. Some of the chemicals tested induced a characteristic karyomorphology that might reflect abnormal cell division. Abnormalities of cell division could be detected in PN and M cells as well as in MN cells. Structure-activity relationships have also been discussed.

We have previously isolated five mutagens in blue rayon-adsorbed substances from water at a site below sewage plants in the Nishitakase River, in Kyoto, Japan, and identified two of them as 2-phenylbenzotriazole derivatives, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). In the present study, we collected adsorbed materials on blue cotton (3 kg x 9 times) at the same location, and isolated a sufficient amount (97 microg) of one of the remaining three mutagens other than PBTA-1 and PBTA-2, for structural analysis, by multiple column chromatography. The structure of mutagen, accounting for 12% of the total mutagenicity of the blue rayon-adsorbed substances, was determined to be a PBTA-1 analogue, 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4). PBTA-4 is a potent mutagen, inducing 190,000 and 7,800,000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix. In addition to the water of the Nishitakase River, PBTA-4 was detected in water samples from two rivers that flow through other regions where textile-dyeing industries have been developed. Like other PBTA analogues, PBTA-4 might also be produced from azo dyes during industrial processes in dyeing factories and treatment at sewage plants.

Mutations induced by one of the typical dietary mutagens/carcinogens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), were characterized using gpt delta transgenic mice. This transgenic mouse model has two selection methods to efficiently detect different types of mutations, i.e. 6-thioguanine selection for point mutations and Spi(-) selection for deletions. The mice were fed with a diet containing 400 p.p.m. PhIP for 13 weeks and gpt and Spi(-) mutations were analyzed from the colon, where the highest mutant frequencies were detected. Concerning the types of gpt mutations from PhIP-treated mice, 81% were single base pair substitutions and G:C-->T:A transversions predominated; single base pair deletions at G:C base pairs were also observed. In untreated mice G:C-->A:T transitions predominated and >80% of these events involved 5'-CpG-3' sites. Concerning Spi(-) mutants from PhIP-treated mice, 76% were G:C base pair deletions and more than half of these events occurred in monotonic G or C run sequences. Interestingly, a novel type of frameshift motif, i.e. G:C base pair deletions beside run sequences, was observed. The most frequently observed mutation in this class was the 5'-TTTTTTG-3'-->5'-TTTTTT-3' event. These results suggest that PhIP induces point mutations, such as base substitutions and single base pair deletions, rather than larger deletions in vivo and that run sequences may play an important role in PhIP-induced G:C base pair deletions.

Harman and norharman are widely distributed in the environment and consequently contaminate in domestic waste-water. It has been reported that they have co-mutagenic activity in the presence of non- mutagenic aromatic amines such as aniline and o-toluidine with S9 mix. When these beta-carbolines were treated with sodium hypochiorite under mild conditions, chlorinated derivatives were produced. Among them, 6-chloroharman and 6-chloronorharman showed much more potent co-mutagenic activities than harman and norharman in the presence of o-toluidine toward Salmonella typhimurium TA98 with S9 mix. These results suggest that the chlorination of harman and norharman occurs during disinfection at the sewage plant to produce potent co-mutagens that contaminate river water.

We have previously determined the chemical structures of two 2-phenylbenzotriazole mutagens (PBTA-1 and PBTA-2) in blue cotton-adsorbed material from the Nishitakase River in Kyoto, Japan. In the present study, further analysis of mutagenic substances in the Nikko River, which flows through Aichi Prefecture in Japan, allowed the isolation of a new mutagen. Material (2.2 g) adsorbed on blue cotton (3 kg) at a site below the sewage plant on the Nikko River was purified by various column chromatographies, and a mutagen (120 microg) accounting for 11% of the total mutagenicity was isolated. On the basis of data from UV, mass, and (1)H NMR spectra of the mutagen, the compound was deduced to be a PBTA-1 analogue. As with PBTA-1, the mutagen was able to be synthesized from the azo dye 2-[(2-bromo-4, 6-dinitrophenyl)azo]-4-methoxy-5-[(2-hydroxyethyl)amino]acetanilide by reduction and chlorination. Since all spectra of the mutagen isolated from the river water were the same as those of the synthesized form, the structure was concluded to be 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino -7-bromo-4-chloro-2H-benzotriazole (PBTA-3). PBTA-3 is a potent mutagen, inducing 81 000 and 3 000 000 revertants per microgram of Salmonella typhimurium TA 98 and YG1024 respectively, in the presence of an S9 mix. In addition to its detection in the water of the Nikko River, PBTA-3 was detected in water samples from three other rivers flowing through regions where dyeing industries have been developed. Like PBTA-1 and PBTA-2, PBTA-3 might have also been produced from azo dyes during industrial processes in dyeing factories and/or through treatment at sewage plants.

To clarify the mutagenic potential of nonagricultural surface soil in Japan, 110 soil samples were collected from five geographically different areas between November 1996 and March 1997, and organic extracts of the soil samples were examined by the Ames/Salmonella assay. Most of the soil extracts showed mutagenicity toward both strains TA98 and TA100 in the presence and/or absence of a mammalian metabolic activation system (S9 mix), suggesting that surface soil is largely contaminated with environmental mutagens. Soil samples collected at Hekinan, Kobe, and Osaka were highly mutagenic toward both strains, and their potencies toward TA98 without S9 mix were extremely high, inducing more than 12 000 revertants per gram of soil. On the other hand, soil samples from Muroran showed strong mutagenicity toward TA100 with S9 mix. Furthermore, 1, 3-dinitropyrene (DNP), 1,6-DNP, and 1,8-DNP in soil samples collected at 10 sampling sites in three metropolitan areas were quantified by fluorometric detection of the corresponding diaminopyrene isomers using high-performance liquid chromatography (HPLC). Three DNP isomers were detected in all soil samples, and the amounts of 1,3-, 1,6-, and 1,8-DNP isomers in the soil samples were 12-3270, 14-5587, and 13-6809 pg/g, respectively. The gross amount of three DNP isomers in surface soil collected at Hekinan was more than 10 ng per gram of soil. The highest contribution ratios of DNP isomers to the mutagenicity of soil extracts were observed for the samples collected at Osaka, and the total of the contribution ratios of three DNP isomers was about 50%. These results suggest that surface soil is largely contaminated with mutagenic compounds and that DNP isomers are one class of major mutagenic and carcinogenic compounds contaminating surface soil.

Group-A xeroderma pigmentosum (XPA) gene-deficient mice are defective in nucleotide-excision repair and highly susceptible to ultraviolet-B-, and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA)-induced skin carcinogenesis. In this study, changes of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP)-DNA adduct formations in the liver, colon and lung, as assessed by the 32P-postlabeling method and immunohistochemical analysis, and carcinogenic and/or toxic susceptibility of both sexes of XPA-deficient mice (XPA-/-) to PhIP, which is a carcinogenic heterocyclic amine, was examined. Levels of PhIP-DNA adduct formations in the liver, colon and lung, were almost twice as high in XPA-/- as in wild type mice (XPA+/+) mice, 7 days after a single i.g. administration of PhIP, and their delay in recovery was observed in XPA-/- mice. For the long-term experiment, XPA-/- and XPA+/+ type mice were treated with 80 ppm PhIP in the diet for the first 4 weeks followed by 40 ppm after a 2-week recovery period (long-term experiment I), or 40 ppm PhIP throughout the experiment (long-term experiment II). Severe toxicity, as evidenced by body weight retardation and poor survival, was observed in the PhIP treated XPA-/- mice of both sexes, but not in the XPA+/+. At week 40 the experiments were terminated and histopathological examinations were performed after complete autopsy. Only lymphomas/leukemias were observed as neoplastic lesions, but no significant differences were observed between the groups. As non-neoplastic lesions, degenerating changes, for example in the pancreatic acinar cells, were observed with XPA-/- mice tending to be more sensitive than XPA+/+ mice. The present study demonstrated that PhIP-DNA adduct formations in the liver, colon and lung of XPA-/- mice were demonstrated and their recovery rate was more delayed than XPA+/+ mice, and furthermore, more severe toxicity to PhIP in XPA-deficient mice was observed, but they were not susceptible to PhIP carcinogenicity under the conditions of the experiment.

The 2-phenylbenzotriazole (PBTA)-type water pollutant, 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), has been recently identified in samples from the Nishitakase River in Kyoto, Japan, and shows potent mutagenic activities in Salmonella typhimurium in the presence of a microsomal metabolizing system (S9 mix). In the present study, we conducted the in vitro micronucleus (MN) test on PBTA-2 in the absence and presence of S9 mix in two Chinese hamster cell lines, CHL and V79-MZ. In the MN test, PBTA-2 was weakly positive in CHL cells and strongly positive in V79-MZ cells. Because the positive results were accompanied by a statistically significant increase in the number of polynuclear (PN) and/or mitotic (M) cells, we examined treated cells in metaphase to see if numerical chromosome aberrations were being induced. We found that PBTA-2 induces polyploidy in both CHL and V79-MZ cells. A detailed analysis of MN preparations showed that in CHL cells, PBTA-2 predominantly induces equal-sized binucleated cells. Rhodamine phalloidin staining revealed that PBTA-2 causes actin filament abnormalities in both cell lines similar to those caused by cytochalasin B. Cytochalasin B induced PN cells predominantly and dose dependently, and almost all the cells were equal-sized and binucleate. The results suggest that PBTA-2 has cytochalasin B-mimetic activity, although agents affecting actin filaments, such as cytochalasins, phallotoxins and chloropeptide, have been derived only from molds so far. This study also suggests that our MN test protocol may be used to identify chemicals that have cytochalasin B-mimetic activity as well as those that induce numerical aberrations.

Previous studies have identified two potent aromatic amine mutagens in the Nishitakase River, a tributary of the Yodo River, which serves as the main drinking water supply for the Osaka area in Japan. The two potent mutagens are 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). PBTA-1 and PBTA-2 are presumed to be formed from azo dyes discharged in a reduced form from dye factories to sewage treatment plants where they become chlorinated and are then discharged into the river. PBTA-1 and PBTA-2 account for 21% and 17% of the mutagenic activity of the Nishitakase River, respectively. Here we determined the mutation spectra induced by these two mutagens in TA98, TA100, and TA104 at 30-35, 8-10, and 2x, respectively, above the background. In TA98, the PBTA compounds produced identical mutation spectra, with 100% of the revertants containing the hotspot 2-base deletion of CG within the (CG)(4) sequence. In TA100, 73% of the revertants were GC-->TA transversions, with most of the remaining being GC-->AT transitions; the spectra produced by the two compounds in TA100 were not significantly different (p=0.8). In TA104, as in TA100, the majority (83%-87%) of the revertants were GC-->TA transversions, with most of the remaining revertants (11%-13%) being AT-->TA transversions. Thus, 83%-87% of the mutations induced by the PBTA compounds in TA104 were at G/C sites. The mutation spectra produced by the two compounds in TA104 were not significantly different (p0.08). PBTA-1 and PBTA-2 are structurally similar and have similar mutagenic potencies and mutation spectra in the respective strains. The mutation spectra produced by the PBTA compounds (100% hotspot deletion in TA98 and primarily GC-->TA transversions in TA100 and TA104) are similar to those produced by other potent aromatic amines, which is the class of compounds from which the PBTA mutagens derive.

Gender differences and organ specificity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis were examined with the new gptdelta transgenic mouse (T. Nohmi, M. Katoh, H. Suzuki, M. Matsui, M. Yamada, M. Watanabe, M. Suzuki, N. Horiya, O. Ueda, T. Shibuya, H. Ikeda, T. Sofuni, A new transgenic mouse mutagenesis test system using Spi-and 6-thioguanine selections (Environ. Mol. Mutagen. 28 (1996) 465-470). In this mouse model, two distinct selections are employed to efficiently detect different types of mutations, i.e 6-thioguanine (6-TG) selection for point mutations and Spi-selection for deletions, respectively. In both selections, the highest mutant frequencies were observed in colon, followed by in spleen and liver. No increases in mutations were observed in testis, brain and bone marrow in PhIP-treated male mice. No significant differences in 6-TG and Spi- mutant frequencies were observed in colon and liver between male and female treated mice. The correlation between PhIP-induced mutagenesis and carcinogenesis in colon is discussed.

The levels of two aromatic amine mutagens, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-a mino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), were quantitatively analyzed in the Yodo River system in Japan. The river water samples were collected at nine sampling sites from the Yodo River system twice or three times between May and July in 1997. PBTA-1 and PBTA-2 in the river water samples were concentrated on blue rayon columns, partially purified by high-performance liquid chromatography (HPLC) on reverse-phase columns, then quantified by HPLC with an electrochemical detector. The amounts of PBTA-1 and PBTA-2 in the water samples were < 0.01-1.91 and < 0.01-2.25 ng/L, respectively. High levels of PBTA-1 and PBTA-2 were detected in the samples collected within 4 km downstream of two sewage plants, which are located along the banks of the Nishitakase River, a tributary of the Yodo River system, and these samples showed stronger mutagenicity in Salmonella typhimurium YG1024 with S9 mix than the other water samples. On the other hand, the river water samples from upstream of the sewage plant were weakly or not mutagenic and PBTA-1 and PBTA-2 were not detected. These results confirmed that a major source of PBTA-1 and PBTA-2 in the Yodo River system is effluent from the sewage plants and that discharged mutagens, including PBTA-1 and PBTA-2, are diluted and/or decomposed while moving down the Yodo River system.

Co-mutagenic beta-carbolines, such as norharman and harman, were quantified in mainstream and sidestream smoke condensates of six Japanese brands of cigarettes, and also in 13 kinds of cooked foods, using a combination of blue cotton treatment and HPLC. Norharman and harman were detected in all the cigarette smoke condensate samples. Their levels in the mainstream smoke case were 900-4240 ng per cigarette for norharman, and 360-2240 ng for harman, and in sidestream smoke, 4130-8990 ng for norharman and 2100-3000 ng for harman. These beta-carbolines were also found to be present in all the cooked food samples, at levels of 2.39-795 ng for norharman and 0.62-377 ng for harman per gram of cooked food. The observed concentrations are much higher than those found for mutagenic and carcinogenic heterocyclic amines (HCAs), suggesting that humans are exposed to norharman and harman in daily life to a larger extent than to HCAs.

Aberrant crypt foci (ACF) are generally considered to be preneoplastic lesions for colon cancer. To assess their induction by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a colon carcinogen, we performed a sequential study of ACF morphology and localization. F344 male rats were given PhIP, and methylene blue-stained colon epithelium and isolated crypts were analyzed at weeks 12, 25, 50, and 75. Each crypt was classified into 2 groups, "single" with round bottoms and "bifurcating" displaying V-shaped clefts (indicating proliferation). In combination with the number of crypts in an ACF, this classification was a good indicator for the generation of ACF in line with the fission mechanism of growth. Increasing numbers of crypts in ACF through weeks 12 to 75 and decreased percentages of ACF with bifurcating crypts at the late time points indicated that proliferation of crypts occurs predominantly during the early stages. The distribution pattern showed a significant shift (P < 0.000005) from the distal to the proximal part of the large intestine between weeks 25 and 50. Adenocarcinomas were first found to develop at week 50 in the ascending colon and cecum where bifurcating crypts were generally lacking at weeks 12 and 25. These data suggest the existence of (1) proliferating ACF which contains bifurcating crypt(s) and (2) quiescent or senescent ACF which consists of only single crypts.

A mutagen, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]5-ami no-7-bromo-4-chloro-2H-benzotriiazole (PBTA-1), isolated from water of the Nishitakase River in Kyoto exhibits potent mutagenic activity in Salmonella typhimurium TA98 with S9 mix and has characteristic moieties, including bromo, chloro, acetylamino, bis(2-methoxyethyl)amino and primary amino groups on a 2-phenylbenzotriazole skeleton. The mutagenicities of PBTA-1, its congeners and five related 2-phenylbenzotriazoles were examined in S. typhimurium TA98 with S9 mix in order to elucidate the structure-activity relationships. The data obtained suggest that a primary amino group plays an essential role in the mutagenic activity as do aromatic amines including heterocyclic amines in cooked foods. The effect of planarity of the 2-phenylbenzotriazole ring was significant, and in addition, halogen groups of PBTA-1 influenced the enhancement of the mutagenic activity., (Copyright 1999 Elsevier Science B.V.)

In order to quantify 1,3-dinitropyrene (DNP), 1,6-DNP and 1,8-DNP in soil, we developed an efficient clean-up procedure and a sensitive determination method using fluorescence detection. DNP isomers were efficiently cleaned by three stages of fractionation, i.e., a silica gel open column chromatography using stepwise elution and two further purification steps by high-performance liquid chromatography (PPLC) using a monomeric-type octadecylsilyl (ODS) column and a polymeric-type ODS column. The recoveries of DNPs during the whole clean-up process were 94% or more. The fraction corresponding to DNPs was injected into an analytical polymeric-type ODS column for HPLC to separate DNP isomers. The effluent from the analytical ODS column was directly introduced to a catalyst column, which was packed with 5 microns alumina coated with platinum and rhodium (Pt-Rh), in order to reduce DNPs to diamino compounds, and then the fluorescence of diaminopyrenes was detected. The immediate detection of diaminopyrene isomers after on-line reduction afforded a sensitive detection of DNP isomers. The detection limits for DNPs were in the range of 0.7 to 4 pg. These developed methods were applied to four soil samples collected at parks in residential areas.

The organic extracts of soil collected at parks in residential areas in Osaka and neighboring cities in the Kansai area, Japan, showed mutagenicity in Salmonella typhimurium strain TA98 in the presence or absence of a mammalian metabolic activation system (S9 mix). The soil extracts from Ibaraki and two different sites in Osaka, i.e., Sumiyoshi-ku and Minato-ku, were mutagenic in strain TA100 as well as in strain TA98. Direct-acting mutagenicity of soil extracts from Sumiyoshi-ku and Minato-ku toward strain TA98 were 66 or more times higher than that of the other cities. Both extracts exerted stronger mutagenicity in strains YG1021 and YG1024 than TA98 and TA100, and the potency was especially high in strain YG1024: Sumiyoshi-ku, 153 000 revertants/g of soil; and Minato-ku, 246 000 revertants/g of soil. Two mutagenic compounds (I and II) were isolated from the Soxhlet extract of soil from the park in Sumiyoshi-ku by repetitive separation using normal-phase and reversed-phase column chromatography. By comparing the mass and UV spectra and retention times for HPLC on two individual ODS columns of compounds I and II with those of authentic chemicals, we identified these two compounds as 1,6- and 1,8-dinitropyrene (DNPy) isomers. Amounts of DNPy isomers in soil from Sumiyoshi-ku and Minato-ku were 1.7-2.2 ng/g. Forty-three percent and 40% of the mutagenicity of soil from Sumiyoshi-ku and Minato-ku could be attributed to these DNPy isomers, respectively.

Norharman (9H-pyrido[3,4-b]indole), widely distributed in our environment, including cigarette smoke and cooked foodstuffs, is not mutagenic to Salmonella strains, but becomes mutagenic to S.typhimurium TA98 and YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline and o-toluidine. To elucidate the mechanisms of co-mutagenicity, we tried to isolate the mutagen(s) produced by a reaction between norharman and aniline with S9 mix. By HPLC purification, two mutagenic compounds (I and II), one (I) showing mutagenicity with and the other (II) without S9 mix, were isolated. The structure of compound I was deduced to be a coupled compound of norharman and aniline, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman), by a variety of spectrometry techniques and this was confirmed by its chemical synthesis. The mutagenic activity of this novel heterocyclic amine was tested using the pre-incubation method and was found to induce 187,000 revertants in TA98 and 1,783,000 revertants in YG1024 per microg with S9 mix. Compound II was shown to be hydroxyaminophenylnorharman. Formation of the same DNA adducts was observed in YG1024 when aminophenylnorharman or a mixture of norharman plus aniline was incubated with S9 mix. The hydroxyamino derivative also yielded the same DNA adducts in YG1024. Thus, the appearance of mutagenicity by norharman with aniline in the presence of S9 mix suggests that the coupled mutagenic compound, aminophenylnorharman, is formed from norharman and aniline, then converted to the hydroxyamino derivative and forms DNA adducts to induce mutations in TA98 and YG1024.

We previously isolated five mutagens, compounds I-V, in blue rayon-adsorbed materials from the Nishitakase River in Kyoto. The chemical structure of compound I, a major mutagen that accounted for 21% of the total mutagenicity, was determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1). Compound II was also a major mutagen and accounted for 17% of the total mutagenicity. In this study, a large quantity (1.2 mg) of compound II was isolated from adsorbate to 27 kg of blue cotton, and its UV, mass, and 1H NMR spectra were analyzed. On the basis of the spectral data, compound II was deduced to be the PBTA-1 analogue 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). As with PBTA-1, PBTA-2 was synthesized from an azo dye by reduction and chlorination. Since all of the spectra of PBTA-2 coincided with those of compound II obtained from river water, compound II was concluded to be PBTA-2. PBTA-2 is a newly identified potent mutagen, which induces 93 000 and 3 200 000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix. Like PBTA-1, PBTA-2 may also be produced from an azo dye during industrial processes in dyeing factories and treatment at sewage plants.