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Monocyclic aromatic amines, o-toluidine (o-Tol) and its structural analog o-anisidine (o-Ans), are IARC Group 1 and Group 2A urinary bladder carcinogens, respectively, and are involved in metabolic activation and DNA damage. Our recent study revealed that 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD), a p-semidine-type homodimer of o-Tol, was detected and identified in an in vitro reaction of o-Tol with S9 mix and in vivo urinary samples of o-Tol-exposed rats. Potent mutagenic, genotoxic, and cytotoxic activities were reported with MMBD, suggesting its involvement in urinary bladder carcinogenesis. However, it remains unknown whether o-Ans is converted to active metabolites to induce DNA damage in a similar manner as o-Tol. In this study, we report that a novel o-Ans metabolite, 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), a dimer by head-to-tail binding (p-semidine form), was for the first time identified in o-Ans-exposed rat urine. MxMxBD induced a stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains and potent genotoxicity and cytotoxicity in human bladder carcinoma T24 cells compared with o-Ans. These results suggest that MxMxBD may to some extent contribute toward urinary bladder carcinogenesis. In addition to homodimerization, such as MxMxBD, heterodimerizations were observed when o-Ans was coincubated with o-Tol or aniline (Ani) in in vitro reactions with S9 mix. This study highlights the important consideration of homodimerizations and heterodimerizations of monocyclic aromatic amines, including o-Ans, o-Tol, and Ani, in the evaluation of the combined exposure risk of bladder carcinogenesis.

o-Toluidine (o-Tol), a monocyclic aromatic amine, causes bladder cancer in humans and experimental animals and is therefore classified as a Group 1 carcinogen (IARC) in which the carcinogenicity of o-Tol is involved in metabolic activation, DNA damage, and DNA adduct formation. In the DNA adduct formation mechanism, o-Tol is metabolized by N-hydroxylation, N-acetoxylation, and then deacetoxylation to produce an electrophilic nitrenium ion, which is able to bind to a DNA base, such as dG-C8. Therefore, dG-C8-o-Tol is thought to be a plausible DNA adduct of o-Tol exposure. However, direct detection of dG-C8-o-Tol in biological samples has not been reported yet. Here, we show that a novel o-Tol metabolite, 2-methyl-N1-(2-methylphenyl)benzene-1,4-diamine (MMBD), a dimer by head-to-tail binding, was identified for the first time in o-Tol-exposed rat urine. MMBD was also detected in a reaction of o-Tol and S9 mix, indicating the formation was catalyzed by an enzymatic reaction. Moreover, MMBD showed a potent stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains,and cytotoxicity in human bladder carcinoma T24 cells and human spleen lymphoblastoid TK6 cells compared with o-Tol. Furthermore, a DNA adduct (m/z 478.1) corresponding to dG-MMBD was detected in the reaction of calf thymus DNA with rat urine containing MMBD, and also in hepatic DNA of rats treated with o-Tol. These results therefore suggested that o-Tol-induced bladder carcinogenesis could be at least partly attributed to MMBD formation. The possible dimerization of monocyclic aromatic amines should be considered in the evaluation of the risk of bladder carcinogenesis after exposure.

A kind of N-nitrosobile acid conjugate, N-nitrosotaurocholic acid (NO-TCA), was incubated with calf thymus DNA, and formation of an adduct was detected by the 32P-postlabeling method under nuclease P1 conditions. To examine the nucleotides containing the adduct from NO-TCA, each of 2'-deoxyribonucleotide 3'-monophosphates (3'-dAp, 3'-dGp, 3'-dCp, or 3'-Tp) was incubated with NO-TCA. The same adduct spot was detected in the reaction of NO-TCA with 3'-dCp. The structure of this adduct was determined to be 3-ethanesulfonic acid-dC by several spectrometry techniques. Moreover, bulky adducts containing bile acid moiety were also produced from the reaction of NO-TCA with 3'-dCp and 3'-dAp. From comparison with spectral data for authentic compounds, these adducts were concluded to be N4-cholyl-dC and N6-cholyl-dA. N4-Cholyl-dC and N6-cholyl-dA were also detected in calf thymus DNA treated with NO-TCA. In addition, 3-ethanesulfonic acid-dC and N4-deoxycholyl-dC were found to be produced from N-nitrosotaurodeoxycholic acid (NO-TDCA) with dC. NO-TCA and NO-TDCA induced mutations in Salmonella typhimurium TA100 but not in TA98. Mutational spectrum analysis revealed that NO-TCA induced G to A transitions predominantly. When NO-TCA (250 mg/kg) was singly administered to male Wistar rats by gavage, both ethanesulfonic acid-dC and N4-cholyl-dC could be detected in the glandular stomach and colon. The levels of ethanesulfonic acid-dC were 0.22-0.29 per 10(6) nucleotides, but values for N4-cholyl-dC were about 500-fold lower. These observations suggest that N-nitroso bile acid conjugates, NO-TCA and NO-TDCA, may induce G to A base substitutions in genes via DNA adduct formation, producing ethanesulfonic acid- and/or (deoxy)cholic acid-DNA and, therefore, may be related to human carcinogenesis as endogenous mutagens.

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.

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.

Two estrogen sulfates, pyridinium 3-methoxyestra-1,3, 5(10)-trien-6alpha-yl sulfate (3MeE-6alpha-S) and its 6beta-isomer (3MeE-6beta-S), synthesized as model compounds to demonstrate the carcinogenesis of estrogen, were found to react with calf thymus DNA to produce steroid-modified DNA adducts. Digestion of the DNA by nuclease P1 and phosphodiesterase I followed by alkaline phosphatase gave a deoxyribonucleoside fraction, of which N2-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyguanosine, N2-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyguanosine, N6-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyadenosine, and N6-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyadenosine (identified as a base adduct) were identified using HPLC by comparing them with authentic specimens prepared by reacting dG and dA with both sulfates. No steroid-dC adduct was detected in the digestion products of the DNA adduct, although dC reacted with the sulfates to form N4-[3-methoxyestra-1,3,5(10)-trien-6beta-yl]deoxycytidine. These results mean that estrogen 6-sulfate has an ability to modify DNA via the amino group of a guanine or adenine residue in DNA. The present studies imply that a sequential metabolism (hydroxylation and sulfation) at the C6-position of the estrogen molecule causes damage to DNA.

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.

Among five mutagenic compounds isolated from water samples, taken at sites below the sewage plants of the Nishitakase River in Kyoto, Japan, the structure of compound I has been determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1). Since this novel aromatic amine mutagen has characteristic substituents in its molecule, it is postulated that the azo dye, 2-[(2-bromo-4, 6-dinitrophenyl)azo]-4-methoxy-5-[bis(2-methoxyethyl)amino]acetoanili de (AZO DYE-1), used as an industrial material, is converted to the corresponding 2-phenylbenzotriazole derivative with a reducing reagent and subsequently to PBTA-1 by chlorination. In fact, AZO DYE-1 changed to the dechlorinated derivative of PBTA-1 (deClPBTA-1) on treatment with sodium hydrosulfite, and this reacted with sodium hypochlorite to produce PBTA-1. Moreover, the presence of deClPBTA-1 was confirmed in a river water sample, along with PBTA-1. PBTA-1 showed potent mutagenic activities in Salmonella typhimurium TA98 and YG1024, inducing 88 000 and 3 000 000 revertants, respectively, per microg, with S9 mix. deClPBTA-1 was also mutagenic, but less potent. From these observations, it is suggested that PBTA-1 is produced from AZO DYE-1 through deClPBTA-1, during industrial processes at dyeing factories and the treatment of wastewater at sewage plants.

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.

Pierisin-1 is a potent apoptosis-inducing protein found in the pupal extract of the cabbage white butterfly. Pierisin-1 catalyzes the mono(ADP-ribosyl)ation of the 2'-deoxyguanosine residue and produces a bulky adduct, N2-(ADP-ribos-1-yl)-2'-deoxyguanosine (N2-ADPR-dG) in DNA. Here, we examined the involvement of the nucleotide excision repair (NER) system in the removal of N2-ADPR-dG in Escherichia coli (E. coli) and human cells. The results of mobility shift gel electrophoresis assays using a 50-mer oligodeoxynucleotide containing a single N2-ADPR-dG showed that E. coli UvrAB proteins bound to the N2-ADPR-dG in vitro. Incubation of the adducted oligodeoxynucleotides with UvrABC resulted in the incision of the oligonucleotides in vitro. The results of filter binding and gel mobility shift assays using human XPA protein showed that XPA bound to DNA containing N2-ADPR-dGs in vitro. Finally, we introduced plasmids containing N2-ADPR-dGs into E. coli and human cells. N2-ADPR-adducted plasmids replicated l0 times and 20 times less efficiently in NER-deficient E. coli and human cells than in their wild-type counterparts, respectively. More mutations were induced in the plasmid propagated in NER-deficient cells than that in wild-type human cells. These results indicate the involvement of the NER system in the repair of N2-ADPR-dG in both E. coli and human cells.

Synthesis of 2'-deoxyguanosine-C8 adducts (dG-C8 adducts) with mutagenic/carcinogenic heterocyclic amines (HCAs) was achieved via the Buchwald-Hartwig arylamination reaction. By using tris(dibenzylideneacetone)dipalladium (Pd(2)dba(3)) and 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (xantphos) with a cesium carbonate (Cs(2)CO(3)) base at a reaction temperature of 100 approximately 120 degrees C, we obtained derivatives of dG-C8 adducts with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in 69% approximately 97% yield from the cross-coupling of an 8-bromodeoxyguanosine derivative. In the case of PhIP, it was found that dimethyl sulfoxide (DMSO) was the critical solvent for the arylamination reaction. Subsequent deprotection of the resulting dG-C8 adduct derivatives yielded authentic samples of dG-C8 adducts with HCAs. The dG-C8-PhIP adduct was further converted into a suitably protected phosphoramidite derivative for automated DNA synthesis. Synthesis of oligonucleotides wherein PhIP adducted on each G within a triple G sequence in codon 869 (TCC GGG AAC) of rat Apc genes was performed with a modification in the coupling time and deprotection procedures.

Cholyl adenylate is a putative intermediate for biosynthesis of cholic acid-coenzyme A (CoA) thioester conjugates by acyl-CoA synthetase. Early studies showed the conjugated acid anhydride moiety of cholyl adenylate to be reactive, attacking proteins to form protein-cholic acid adducts. In the present study, to clarify reactions of cholyl adenylate with DNA under physiological conditions, products with nucleosides were analyzed. HPLC-MS analyses indicated cholyl adenylate to primarily attack hydroxy groups of ribose moieties of nucleosides. Moreover, as speculated from UV and MS studies, exocyclic amino groups of 2'-deoxycytidine and 2'-deoxyadenosine were found to serve as targets of cholyl adenylate; the corresponding cholic amides, N4-cholyl-2'-deoxycytidine and N6-cholyl-2'-deoxyadenosine, were formed at yields of 0.32 and 0.06%, respectively. Structures of these base modified adducts were confirmed by direct comparison with synthetic compounds obtained from coupling reactions of cholic acid with each nucleoside in the presence of dicyclohexylcarbodiimide in pyridine at 70 degrees C. N4-Cholyl-2'-deoxycytidine was also obtained at a level of 1.6 adducts per 10(5) nucleosides from enzymatic hydrolysates of calf thymus DNA reacted with cholyl adenylate. These results suggest that cholyl adenylate, released from CoA synthetase, may have some possibility as a DNA modifier in vivo.

Pierisin-1, an ADP-ribosylating toxin derived from the cabbage butterfly, Pieris rapae, induces apoptosis in various mammalian cell lines. We recently reported that the target for ADP ribosylation by pierisin-1 is the 2'-deoxyguanosine residue in DNA. To examine whether pierisin-1 would induce mutations in mammalian cell genes, we conducted a mutational analysis for the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in pierisin-1-treated Chinese hamster lung (CHL) cells. N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine was detected by the (32)P-postlabeling method in CHL cells after treatment with pierisin-1 at doses of 2-32 ng/mL; adduct levels were 1.1-12.0 per 10(6) nucleotides. Pierisin-1 induced mutations in the HPRT gene dose-dependently, and the frequency was 38 times higher than the control, at a dose of 32 ng/mL. To confirm that mono(ADP-ribosyl)ated dG itself leads to mutations, the pierisin-1-treated DNA of plasmid pMY189 bearing the supF gene was used for mutational analysis. The mutation frequency of the supF gene treated with 2-8 micro g/mL of pierisin-1 was 17-40-fold the control value. Mutation spectrum analysis showed that single base substitutions dominated in both HPRT and supF genes. Among these, transversions were predominant, and more than 70% of the base substitutions occurred at G:C base pairs in both genes. The most frequent mutations were G:C to C:G, followed by G:C to T:A in HPRT gene, whereas G:C to T:A transversions dominated in the supF gene. Our results indicate that pierisin-1 produced N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine and this guanine-adduct could lead to mutations in the HPRT and supF genes. These findings could provide very useful information for understanding the biological significance of pierisin-1.

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.

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.

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.

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.

Water samples from the Nishitakase River in Kyoto, Japan, especially taken at sites below sewage plants, show significantly high mutagenicity in the Ames test. In the present study, mutagens in the river water were adsorbed to 24 g of blue rayon, extracted, and separated by HPLC on ODS columns. Five mutagenic compounds (I-V) were isolated, and they accounted for 21%, 17%, 11%, 12%, and 6%, respectively, of the total mutagenicity of the blue rayon-adsorbed materials. With compound I obtained from adsorbate to 24 g of blue rayon as a marker, a large quantity (1.1 mg) of mutagenic compound I was isolated by Sephadex LH-20 column chromatography and HPLC on ODS columns from material adsorbed to 27 kg of blue cotton. X-ray crystal analysis was carried out with the debrominated derivative of compound I. Based on this X-ray crystallography data and the UV, mass, and 1H-NMR spectra of both the derivative and compound I, the structure of compound I was determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino - 7-bromo-4-chloro-2H-benzotriazole (PBTA-1). PBTA-1 is a newly identified potent mutagen, inducing 1,200,000 revertants of Salmonella typhimurium YG1024 per microgram in the presence of S9 mix.