Identification of a new mutagen, 4,4'-diamino-3,3'-dichloro-5-nitrobiphenyl, in river water flowing through an industrial area in Wakayama, Japan

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.     

Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water

Blue cotton, bearing a covalently bound copper-phthalocyanine derivative capable of adsorbing polycyclic aromatic hydrocarbons (PAHs) over 3 rings, was applied to recover mutagens from the Katsura River which is a tributary of the Yodo River. The Ames Salmonella/microsome assay with TA98 and TA100 of the blue cotton concentrate recovered from the river water demonstrated indirect mutagenicity toward TA98. The subfractions separated by Sephadex G-25 gel chromatography also showed direct mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98; this activity was greatly increased by the addition of S9 mix, especially in YG1024. However, these subfractions were less mutagenic with TA98NR or TA98/1,8-DNP6, regardless of whether S9 mix was present or not. The behaviors of these mutagenic activities therefore suggested that frameshift mutagens of both directly mutagenic nitroarenes and indirectly mutagenic aminoarenes were present in the blue cotton concentrate from the river water.     

Seasonal fluctuation of the mutagenicity of river water in Fukui,Japan, and the contribution of 2-phenylbenzotriazole-type mutagens

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.     

Application of Salmonella strains with altered nitroreductase and O-acetyltransferase activities to the evaluation of the mutagenicity of airborne particles

The Ames test was applied to evaluation of the mutagenicity of month's samples of airborne particles from the center of Wroc?aw (SW Poland) collected in August and December 1997. The strains used for the study were TA 98, TA 100 and their derivatives: TA 98 NR, YG 1021, YG 1024, YG 1026, YG 1029, YG 1041, YG 1042. Both studied samples were mutagenic for almost all tested strains, with the exception of the August sample which did not influence the strain TA 100 without the metabolic activation with the S9 fraction. The December sample exhibited higher genotoxic activity than the August sample. Mutagenicity ratios of the strains with reduced nitroreductase and O-acetyltransferase activities were higher, and of the strain without the nitroreductase--lower than those of the parent strains. This indicates that nitro and amino derivatives of PAHs are responsible for the significant proportion of total mutagenicity of the studied samples of particulates. Metabolic activation with the S9 fraction caused the increase of the mutagenic activity of the samples, which indicates the presence of promutagens. The GC-MS analysis revealed the presence of known indirect mutagens from the PAHs group.     

Levels and behavior of 2-phenylbenzotoriazole-type mutagens in the effluent of a sewage treatment plant

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.     

Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers

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.     

Mutagenicity of native cigarette mainstream smoke and its gas/vapour phase by use of different tester strains and cigarettes in a modified Ames assay

The "Bacterial Reverse Mutation Assay" is generally accepted to analyse the genotoxic capacity of single compounds or complex mixtures such as cigarette-smoke condensates. With an adapted and modified Ames assay, the mutagenicity of native cigarette mainstream whole smoke (WS) and its gas/vapour phase (GVP) was studied. The bacteria were directly exposed to the smoke in a CULTEX1 system closely connected to a smoking robot (VC10). A variety of standard tester strains (TA98, TA100, TA1535, TA1537, TA1538, TA102, WP2uvrApKM101) and descendants of TA98 (YG1021, YG1024, YG1041) and TA100 (YG1026, YG1029 and YG1042) were exposed to whole and filtered smoke of the research cigarette K2R4F to find the most sensitive strains for analysing the mutagenic activity of these test atmospheres. Mutagenicity of WS was detected by TA98, TA100 and their YG descendant strains as well as by WP2uvrApKM101 in the presence of S9 mix. The GVP induced a mutagenic signal in TA100, YG1029 and YG1042 and WP2uvrApKM101 only in the absence of S9 mix. To detect mutagenicity in WS the presence of the plasmid pKM101 is required and a frame-shift mutation is more effective than a missense mutation. To detect mutagenicity in GVP, the presence of the plasmid pKM101 and a missense mutation are required. The differentiating capacity of this modified Ames assay was demonstrated by exposing strain TA98 to WS and TA100 to the GVP of cigarettes with different tar content. The mutagenic activity of WS and the GVP increased with rising tar content of the cigarettes with two exceptions in WS. Thus, the concept of tar content alone is misleading and does not reflect the mutagenic activity of a cigarette.     

Blue rayon-anchored technique/Salmonella microsome microsuspension assay as a tool to monitor for genotoxic polycyclic compounds in Santos estuary

The most important harbor of Brazil is located in Santos Estuary. In the 1970s, this area was one of the major examples of coastal degradation and although the quality of the environment has improved, the sediment is still contaminated with polycyclic aromatic hydrocarbons (PAHs) and mutagenic activity. Because of sediment dredging and consequently contaminants resuspension, it is useful to have reliable methods to monitor the water quality. Considering that blue rayon (BR) has been successfully used in evaluation of mutagenicity and PAHs content the objective of this work was to verify the applicability and adapt the methodology to monitor the water for mutagenic activity using the BR associated with the Salmonella assay. Analysis of three sites with different levels of contamination was performed using a modification of the BR hanging method denominated in this work BR anchored technique. The microsuspension protocol of the Salmonella/microsome assay was employed with the strain YG1041. The water from the site 1 the most contaminated and under influence of the steel mill discharge presented the highest potency reaching 36,000 revertants/g of BR with S9. Sites 2 and 3 showed less mutagenicity than site 1 with values approximately 1000 revertants/g of BR. We conclude that the BR anchored technique associated with Salmonella assay using YG1041 is a reliable alternative to monitor estuarine waters, especially in regions where sediment resuspension or acute pollution episodes can occur.     

Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan

We previously determined the chemical structures of four 2-phenylbenzotriazole mutagens (PBTA-1, -2, -3 and -4) in blue rayon-adsorbed material from the Nishitakase River in Kyoto prefecture and the Nikko River in Aichi prefecture in Japan. On the basis of a synthesis study, these four PBTA derivatives were deduced to have originated from corresponding dinitrophenylazo dyes by reduction and chlorination. 2-[(2-Bromo-4,6-dinitrophenyl)azo]-5-[bis(2-acetoxyethyl) amino]-4-methoxyacetanilide (Color Index Name, Disperse Blue 79:1; CAS Registry Number, 75497-74-4) is a very common dinitrophenylazo dye used in textile dyeing factories. In the present study, we synthesized 2-[4-[bis(2-acetoxyethyl)amino]-2-(acetylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-5) from Disperse Blue 79:1 by reduction with sodium hydrosulfite and subsequent chlorination with sodium hypochlorite. On hydrolysis of PBTA-5 with alkali, 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) was obtained. Both PBTA-5 and -6 were potent mutagens, inducing 723,000 revertants and 485,000 revertants per microgram of Salmonella typhimurium YG1024, respectively, in the presence of S9 mix. To clarify whether PBTA-5 and -6 exist in the environment, water samples were collected from five rivers flowing through regions where textile dyeing industries are developed. PBTA-6 was detected at levels of 3–134 ng/g blue rayon in all water samples that were examined. On the other hand, the amount of PBTA-5 in the samples was less than the detection limit.     

Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan.

We previously determined the chemical structures of four 2-phenylbenzotriazole mutagens (PBTA-1, -2, -3 and -4) in blue rayon-adsorbed material from the Nishitakase River in Kyoto prefecture and the Nikko River in Aichi prefecture in Japan. On the basis of a synthesis study, these four PBTA derivatives were deduced to have originated from corresponding dinitrophenylazo dyes by reduction and chlorination. 2-[(2-Bromo-4,6-dinitrophenyl)azo]-5-[bis(2-acetoxyethyl) amino]-4-methoxyacetanilide (Color Index Name, Disperse Blue 79:1; CAS Registry Number, 75497-74-4) is a very common dinitrophenylazo dye used in textile dyeing factories. In the present study, we synthesized 2-[4-[bis(2-acetoxyethyl)amino]-2-(acetylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-5) from Disperse Blue 79:1 by reduction with sodium hydrosulfite and subsequent chlorination with sodium hypochlorite. On hydrolysis of PBTA-5 with alkali, 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) was obtained. Both PBTA-5 and -6 were potent mutagens, inducing 723,000 revertants and 485,000 revertants per microgram of Salmonella typhimurium YG1024, respectively, in the presence of S9 mix. To clarify whether PBTA-5 and -6 exist in the environment, water samples were collected from five rivers flowing through regions where textile dyeing industries are developed. PBTA-6 was detected at levels of 3-134 ng/g blue rayon in all water samples that were examined. On the other hand, the amount of PBTA-5 in the samples was less than the detection limit.     

Evaluation of a battery of Salmonella typhimurium tester strains for biomonitoring of mutagenic polycyclic aromatic hydrocarbons, nitroarenes and aromatic amines

Various combinations of Salmonella typhimurium tester strains and S9 mix for bioactivation (TA98+S9 mix, TA98S; YG1041+S9 mix, YG1041S) and strain YG1041 in the absence of S9 mix (YG1041) were used to evaluate the mutagenic activity of eight polycyclic aromatic hydrocarbons (PAHs), seven nitroarenes (NAs) and seven aromatic amines (AAs). Three cigarette smoke extracts and two extracts of smokers' urine (SUE) were also included. Urinary mutagenicity was then determined on 31 individuals, potentially exposed to PAHs, for 0 h, 7 h, 12 h and 24 h. Concentrations of urinary 1-hydroxypyrene (1OHP) and 3-hydroxybenzo[a]pyrene (3OHBaP), the levels of atmospheric pyrene (Py) and benzo[a]pyrene (BaP), and particulate concentrations in air (AP) were also measured. PAHs could be detected by TA98S and YG1041S, with TA98S being more sensitive than YG1041S. While NAs could be detected by all combinations, YG1041 and YG1041S were more sensitive than TA98S. Although both YG1041S and TA98S could detect AAs, YG1041S was more sensitive than TA98S. Cigarette smoke extract contained mutagenic AAs and NAs, but AAs were the only mutagenic compounds detected in the extracts of smokers' urine. The concentrations of 1OHP (7 h and 12 h) were significantly higher than those at 0 h, but no difference could be detected with 3OHBaP. Correlations were found between Py and 1OHP (7 h and 24 h) and between BaP and 3OHBaP concentrations (7 h, 12 h and 24 h). A significantly elevated urinary mutagenicity was detected with YG1041S at 7h in the group of smokers. A good correlation was determined between AP and the test results with TA98S (7 h) and with YG1041 (0 h and 7 h). Urinary 1OHP correlated with the test results with YG1041S (0 h, 7 h and 12 h) while 3OHBaP correlated with those obtained with YG1041S (7 h). Overall, 21/31 individuals were occupationally exposed to AAs, 15/31 individuals were exposed to NAs, and 2/31 were exposed to PAHs as indicated by the Salmonella mutagenicity assay. The urine mutagenicity test was not effective at monitoring occupational exposure to PAHs. However, the correlation with AP implied the presence of unknown mutagenic atmospheric substances that could modulate the urinary mutagenicity.     

Evaluation of nitroreductase and acetyltransferase participation in N-nitrosodiethylamine genotoxicity

N-Nitroso compounds, such as N-nitrosodiethylamine (NDEA), are a versatile group of chemical carcinogens, being suspected to be involved in gastrointestinal tumors in humans. The intestinal microflora can modify a wide range of environmental chemicals either directly or in the course of enterohepatic circulation. Nitroreductases from bacteria seem to have a wide spectrum of substrates, as observed by the reduction of several nitroaromatic compounds, but their capacity to metabolize N-nitroso compounds has not been described. To elucidate the participation of nitroreductase or acetyltransferase enzymes in the mutagenic activity of NDEA, the bacterial (reverse) mutation test was carried out with the strains YG1021 (nitroreductase overexpression), YG1024 (acetyltransferase overexpression), TA98NR (nitroreductase deficient), and TA98DNP6 (acetyltrasferase deficient), and YG1041, which overexpresses both enzymes. The presence of high levels of acetyltransferase may generate toxic compounds that must be eliminated by cellular processes or can lead to cell death, and consequently decrease the mutagenic effect, as can be observed by the comparison of strain TA98DNP6 with the strains TA98 and YG1024. The slope curves for TA98 strain were 0.66 rev/microM (R(2) = 0.51) and 52.8 rev/microM (R(2) = 0.88), in the absence and presence of S9 mix, respectively. For YG1024 strain, the slope curve, in the presence of S9 mix was 6897 rev/microM (R(2) = 0.78). Our data suggest that N-nitroso compounds need to be initially metabolized by enzymes such as cytochromes P450 to induce mutagenicity. Nitroreductase stimulates toxicity, while acetyltransferase stimulates mutagenicity, and nitroreductase can neutralize the mechanism of mutagenicity generating innoccuos compounds, probably by acting on the product generated after NDEA activation.     

Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

Detection of mutagenicity of diphenyl ether herbicides in Salmonella typhimurium YG1026 and YG1021

Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per μg. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per μg. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.     

Identification of 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) as a potent mutagen in river water in Kyoto and Aichi prefectures, Japan

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.     

Mutagenicity of surface soil from residential areas in Kyoto city, Japan, and identification of major mutagens

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.     

Mutagenicity of urine from rats after 1-nitropyrene and 2-nitrofluorene administration using new sensitive Salmonella typhimurium strains YG1012 and YG1024

1-Nitropyrene (1-NP) and 2-nitrofluorene (2-NF), two of the most abundant nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAH) present in combustion products such as diesel engine exhaust, were administered intraperitoneally to rats at a dose of 5 mg per animal. Urine samples, 1-NP and 2-NF were tested in the Ames assay using the newly developed Salmonella typhimurium strains YG1012 and YG1024 (overproducing O-acetyltransferase) and their parent strains TA1538 and TA98. In urine, collected over 3 periods of 24 h after administration, most of the mutagens appeared during the first 24 h. The mutagenicity was found to be a factor 2-30 higher in the YG strains when compared to the TA strains. Addition of S9 mix and rat liver cytosol both with and without beta-glucuronidase increased the mutagenicity of urine samples from 1-NP-treated rats. Addition of beta-glucuronidase revealed that a considerable part of the mutagenic metabolites of 1-NP and 2-NF were excreted as glucuronide conjugates. The increase in mutagenicity of urine samples from 2-NF-treated rats after the addition of rat liver cytosol referred to N,O-acyl transfer as a step in activating 2-NF to strong mutagens. The high sensitivity of the YG tester strains indicated that these strains might be used to explore environments where people are exposed to nitro-PAH, such as work places with diesel emission sources.     

Mutagenicity assay for nitroarenes of air pollutants held in leaves of woody plants.

A new method was developed for the detection of mutagenic nitroarenes held in the leaves of woody plants using a small amount of leaves. This method consists of extraction of mutagen from fresh leaves (15-30 g) by ultrasonication with ethyl acetate and purification by elution with benzene on a silica gel column to remove such inhibitory components for mutagenesis as chlorophyll. The mutation assay uses the new Salmonella typhimurium strains YG1021 (a nitroreductase-overproducing strain of TA98) and YG1024 (an O-acetyltransferase-overproducing strain of TA98), which are very sensitive to some nitroarenes in the absence of S9, and standard strain YG1020 (TA98 containing pBR322-Aps) for comparison. This method was applied to woody plants growing on various sites in the Tokyo metropolitan area and the suburbs. It was shown that the leaves of all woody plants tested contained different amounts of mutagens, probably mutagenic nitroarenes, depending upon their growing sites.     

Mutagenicity of bay-region amino-substituted cyclopenta[a]phenanthrenes and 2- and 5-aminochrysene

The relative mutagenic potentials of 11-amino-16,17-dihydro-15H-cyclopenta[a]phenanthrene, its 17-keto derivative, and 2- and 5-aminochrysene have been compared in Salmonella typhimurium TA98 and TA100 in the presence of a postmitochondrial liver preparation from Aroclor 1254 induced rats. The 11-amino hydrocarbon is a very weak mutagen (0.27 revertants/nmol), whereas the 11-amino-17-ketone is much more active (129 revertants/nmol). 2-Aminochrysene is the most mutagenic arylamine ( approximately 500 revertants/nmol) among these compounds, but its 5-amino isomer is much less active (0.9 revertants/nmol). Possible reasons for these marked differences are suggested.Use of TA98 with over-expressing O-acetyltransferase (YG 1024) and deficient in this enzyme (TA98/l,8-DNP(6)) with the 11-amino-17-ketone and with 5-aminochrysene clearly indicates the importance of this enzyme in their bioactivation, implying oxidation of the amino group to the hydroxylamine in both these compounds.