Major Mutagen Research Articles

Labor- and cost-effective long-read amplicon sequencing using a plasmid analysis service: Application to transposon-inserted alleles in Japanese morning glory.

The sequencing of PCR fragments amplified from specific regions of genomes is a fundamental technique in molecular genetics. Sanger sequencing is commonly used for this analysis; however, amplicon sequencing utilizing next-generation sequencing has become widespread. In addition, long-read amplicon sequencing, using Nanopore or PacBio sequencers to analyze long PCR fragments, has emerged, although it is often more expensive than Sanger sequencing. Recently, low-cost commercial services for full-length plasmid DNA sequencing using Nanopore sequencers have been launched in several countries, including Japan. This study explored the potential of these services to sequence long PCR fragments without the need for cloning into plasmid DNA, as cloning long PCR fragments or blunt-end PCR fragments into plasmid DNA is often challenging. PCR fragments of 4-11 kb, amplified from the DFR-B gene involved in the biosynthesis of anthocyanin, with or without Tpn1 transposons in Japanese morning glory (Ipomoea nil), were circularized using T4 DNA ligase and analyzed as templates. Although some inaccuracies in the length of homopolymer stretches were observed, the remaining sequences were obtained without significant errors. This method could potentially reduce the labor and costs associated with cloning, primer synthesis, and sequence assembly, thus making it a viable option for the analysis of long PCR fragment sequences. Moreover, this study reconfirmed that Tpn1 transposons are major mutagens in I. nil and demonstrated their transposition in the Violet line, a long-used standard in plant physiology.

Intragenomic variability and extended sequence patterns in the mutational signature of ultraviolet light

Mutational signatures can reveal properties of underlying mutational processes and are important when assessing signals of selection in cancer. Here, we describe the sequence characteristics of mutations induced by ultraviolet (UV) light, a major mutagen in several human cancers, in terms of extended (longer than trinucleotide) patterns as well as variability of the signature across chromatin states. Promoter regions display a distinct UV signature with reduced TCG > TTG transitions, and genome-wide mapping of UVB-induced DNA photoproducts (pyrimidine dimers) showed that this may be explained by decreased damage formation at hypomethylated promoter CpG sites. Further, an extended signature model encompassing additional information from longer contextual patterns improves modeling of UV mutations, which may enhance discrimination between drivers and passenger events. Our study presents a refined picture of the UV signature and underscores that the characteristics of a single mutational process may vary across the genome.

The effect of silver nanoparticles on the mutagenic and the genotoxic properties of the urban wastewater liquid sludges.

Nanoparticles are very effective compounds to transform and detoxicate common environmental contaminants. For this reason, crude urban liquid wastewater sludges were treated by silver nanoparticles (Ag-NPs, 100 nm) for 24 h. Both Ag-NPs' treated and untreated sludges were examined for the evaluation if there are possible mutagenic/anti-mutagenic, cytotoxic, and genotoxic/anti-genotoxic effects by Ames and Allium cepa tests. The results were then subjected to statistical analyses by using SPSS software and p < 0.05 was accepted as a significant value. The data obtained from the Ames test showed that while untreated crude liquid sludge had a significant mutagenic effect, Ag-NP-treated one decreased its mutagenicity. Similar effects were also observed in the chromosome aberration-Allium cepa tests. Significant chromosome aberrations observed were C-metaphase, sticky metaphase, sticky anaphase, anaphase bridge, vagrant chromosome, and multipolar anaphases. Both tests demonstrated that silver nanoparticle treatment decreased the major mutagenicity and genotoxicity detected in the liquid wastewater sludges.

Identification of a mutagenic chlorination by-product produced from (E)-1, 3-dichloropropene (a component of nematocide DD) by using high resolution LC/MS and multivariate analysis

The estimated domestic usage of DD (a typical nematocide) has been the greatest among all the agricultural chemicals in Japan. DD is involved in a document which is used to establish tap water quality standard in Japan. The document indicates that DD is potentially detectable in raw water for tap waters. DD in raw water will be treated with chlorine at waterworks, which raises concerns about formation of mutagenic chlorination by-products through the treatment. The objective of this work was to identify a mutagenic chlorination by-product by using high resolution mass spectrometers and multivariate analyses. Mutagenicity of the chlorination by-product was evaluated with the Ames Salmonella mutagenicity assay. (E)-1,3-Dichloropropene (DCP) was used as a model compound of DD. The mutagenicity of chlorine treated model aqueous solutions of DD increased with increasing chlorine dosages up to 3.00 mol-Cl2/mol-DCP. From the chlorine treated aqueous solutions of DD, 1,3-dichloroacetone (DCA) was identified as a major mutagen by a cochromatography with an authentic standard, precursor ion analyses and first-generation product ion analyses with the high resolution mass spectrometers. The mutagenicity of DCA against TA100 strain without microsomal activation was 160,000 net revertant colony/μmol (the toxicity equivalent factor was 0.14). Based on the mutagenicity of the chlorine treated DCP sample, the specific mutagenicity of DCA, and the DCA concentration in the sample, the mutagenic contribution of DCA to the chlorine treated DCP sample was calculated. High contribution (98%) clearly shows that DCA is a major mutagen in the chlorine treated DCP sample. Because the exploration of DCA was performed by using the unrealistic high concentration samples of DCP and chlorine, formation of DCA in the practical concentration samples (0-200 μg-DCP/L, 0.10 or 1.27 mg-Cl2/L) was examined. It was proven that DCA formation reaction takes place in the practical concentrations of DCA and chlorine.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

Identification of methylglyoxal as a major mutagen in wood and bamboo pyroligneous acids.

To identify the major mutagen in pyroligneous acid (PA), 10 wood and 10 bamboo pyroligneous acids were examined using the Ames test in Salmonella typhimurium strains TA100 and TA98. Subsequently, the mutagenic dicarbonyl compounds (DCs), glyoxal, methylglyoxal (MG), and diacetyl in PA were quantified using high-performance liquid chromatography, and the mutagenic contribution ratios for each DC were calculated relative to the mutagenicity of PA. Eighteen samples were positive for mutagens and showed the strongest mutagenicity in TA100 in the absence of S9 mix. MG had the highest mutagenic contribution ratio, and its presence was strongly correlated with the specific mutagenicity of PA. These data indicate that MG is the major mutagen in PA.

Identification and quantitative confirmation of dinitropyrenes and 3-nitrobenzanthrone as major mutagens in contaminated sediments

Polar fractions of a sediment extract of the industrial area of Bitterfeld, Germany, have been subjected for effect-directed identification of mutagens using the Ames fluctuation assay with TA98. Mutagenicity could be well recovered in several secondary and tertiary fractions. Dinitropyrenes and 3-nitrobenzanthrone could be confirmed to contribute great shares of the observed mutagenicity. In addition, a multitude of polar polycyclic aromatic compounds has been tentatively identified in mutagenic fractions including nitro-PAHs, azaarenes, ketones, quinones, hydroxy-compounds, lactones and carboxylic acids although their contribution to mutagenicity could not be quantified due to a lack of standards. Diagnostic Salmonella strains YG1024 and YG1041 were applied to confirm the contribution of nitro-aromatic compounds. We suggest the inclusion of dinitropyrenes and 3-nitrobenzanthrone into sediment monitoring in order to minimize the mutagenic risk to aquatic organisms and to human health.

Chlorination by-products of fenitrothion

The mutagens produced through chemical reaction between chlorine and the insecticide fenitrothion were studied by using a quadrupole GC-MS. The mutagenicity and the mutagen formation potential (MFP) of the identified by-products were evaluated by the Ames assay (preincubation method) using Salmonella typhimurium TA100 without exogenous activation by S9 mix (TA100-S9). Before conducting GC/MS analyses, six compounds were presumed to be produced in chlorinated fenitrothion. These compounds were confirmed to be produced by the GC/MS analyses, but none of them were mutagenic. One of the chlorination by-products, 3-methyl-4-nitrophenol, has 19 times greater MFP than that of fenitrothion. This result suggests that a major mutagen in chlorinated fenitrothion will be produced via a chemical reaction between chlorine and 3-methyl-4-nitrophenol.

Determination of 3,6-dinitrobenzo[e]pyrene in Surface Soil and Airborne Particles, and Its Possible Sources, Diesel Particles and Incinerator Dusts

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP) is an extremely strong bacterial mutagen, and was recently identified in highly mutagenic surface soil samples. In a previous study, a sensitive analytical method was developed using high-performance liquid chromatography (HPLC) and fluorescence detection. In this study, we analyzed 3,6-DNBeP in surface soil, airborne particles, diesel particles, and incinerator dusts using this analytical method to reveal the distribution of 3,6-DNBeP in the environment. 3,6-DNBeP was detected in all surface soil samples, and the mutagenic contribution ratio of 3,6-DNBeP to the mutagenicity of the soil extracts toward Salmonella (S.) typhimurium TA98 was 17.3% on average. A positive correlation was observed between the mutagenicity of surface soil and the amount of 3,6-DNBeP (r = 0.8653). 3,6-DNBeP was detected in airborne particles in the range of 19–76 fg/m 3 .T heparticle-size-distribution ratios of 3,6-DNBeP in 7µm of airborne particles were 13.1%, 13.8%, 37.0%, 19.1%, and 17.0%, respectively. 3,6-DNBeP was detected in diesel particles from general automobiles and industrial forklifts, and incinerator dusts. These results suggested that 3,6-DNBeP was a major mutagen in surface soil, and diesel engines and incinerators were possible sources of 3,6-DNBeP distributed in surface soil and air. This is the first report on the detection of 3,6-DNBeP in diesel particles and incinerator dusts.

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

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

Effect of the GC content of DNA on the distribution of UVB-induced bipyrimidine photoproducts

Solar UV radiation is a major mutagen that damages DNA through the formation of dimeric photoproducts between adjacent thymine and cytosine bases. A major effect of the GC content of the genome is thus anticipated, in particular in prokaryotes where this parameter significantly varies among species. We quantified the formation of UV-induced photolesions within both isolated and cellular DNA of bacteria of different GC content. First, we could unambiguously show the favored formation of cytosine-containing photoproducts with increasing GC content (from 28 to 72%) in isolated DNA. Thymine-thymine cyclobutane dimer was a minor lesion at high GC content. This trend was confirmed by an accurate and quantitative analysis of the photochemical data based on the exact dinucleotide frequencies of the studied genomes. The observation of the effect of the genome composition on the distribution of photoproducts was then confirmed in living cells, using two marine bacteria exhibiting different GC content. Because cytosine-containing photoproducts are highly mutagenic, it may be predicted that species with genomes exhibiting a high GC content are more susceptible to UV-induced mutagenesis.

Tyramine is a major mutagen precursor in soy sauce, being convertible to a mutagen by nitrite.

Tyramine was identified as a new mutagen precursor in Japanese soy sauce, becoming mutagenic after treatment with nitrite under acidic conditions. The mutagenic compound was identified as 4-(2-aminoethyl)-6-diazo-2,4- cyclohexadienone , and its specific mutagenic activity was 112 revertants/micrograms towards Salmonella typhimurium TA100 without S9 mix.

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.

Isolation and identification of non-chlorinated phenylbenzotriazole (non-ClPBTA)-type mutagens in the Ho River in Shizuoka Prefecture, Japan

We previously identified 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2 H-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-2 H-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-2 H-benzotriazole (non-ClPBTA-2), 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-6-amino-4-bromo-2 H-benzotriazole (non-ClPBTA-3), 2-(2-acetylamino-4-amino-5-methoxyphenyl)-6-amino-4-bromo-2 H-benzotriazole (non-ClPBTA-4), and 2-[2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl]-6-amino-4-bromo-2 H-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/μg, 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.

Identification of 1,3,6-Trinitropyrene as a Major Mutagen in Organic Extracts of Surface Soil from Nagoya City, Japan

An organic extract from surface soil collected at a park in Nagoya, Aichi prefecture, which showed extremely high mutagenicity in Salmonella typhimurium TA98 in the absence of a mammalian metabolic system (S9 mix), was investigated to identify the major mutagens. A potent bacterial mutagen was isolated from the organic extract (1.03 g) of the soil sample (2.1 kg) by column chromatography. On the basis of mass spectra, the mutagen, which accounted for 8.8% of the mutagenicity of the soil extract, was thought to be a trinitrated polycyclic aromatic hydrocarbon with a molecular weight of m/z 337. The mutagen was synthesized from pyrene by nitration with nitric acid and was identified as 1,3,6-trinitropyrene (1,3,6-TNP) based on its 1H NMR spectrum. The mutagenicity of 1,3,6-TNP in an Ames assay using S. typhimurium was extremely high, in that it induced 65,500 revertants/nmol in TA98, without S9 mix. This level of activity was slightly lower than that of 1,6-dinitropyrene (DNP), 129,800 revertants/nmol, or 1,8-DNP, 217,000 revertants/nmol, but was greater than that of 1,3-DNP, 4,260 revertants/nmol. These results indicate that 1,3,6-TNP was one of the major mutagens in surface soil collected at a park in Nagoya city, and this is the first report on the detection of 1,3,6-TNP in surface soil.

TLC Fractionation and Characterization of Ames Mutagenic Substances in Chlorine-treated 4-methylphenol Solution in the Presence of Bromide Ion

The diethyl ether extracts from aqueous 4-methylphenol solutions in the presence of bromide ion after treatment with chlorine were mutagenic to the Ames salmonella test strain TA100 in the absence of rat liver homogenate. Gas chromatography/mass spectrometry (GC/MS) showed the occurrence of halogenated products in the extracts: bromo-4-methylphenols, bromo-4-methylquinones and brominated 4-methylphenol dimers. The diethyl ether extracts were fractionated into several fractions by polyamide thin-layer chromatography (TLC) . The fractionated components were examined for mutagenicity by means of Ames assays, and were identified by GC/MS. TLC fraction of the extracts revealed that two components present in the extracts are mutagenic. GC/MS analysis indicated the presence of 2, 6-dibromo-4-hydroxy-4-methylcyclohexa-2, 5-lien-1-one (2, 6-dibromotolquinol) as the major mutagen and brominated 4-methylphenol dimers as minor mutagenic compounds in the chlorinated 4-methylphenol solutions containing bromide ion. Production of 2, 6-dibromotolquinol in water was found to be dependent on the equivalents of chlorine per mole of compound and the reaction pH.

Detection of a Novel Mutagen, 3,6-Dinitrobenzo[e]pyrene, as a Major Contaminant in Surface Soil in Osaka and Aichi Prefectures, Japan

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.

Mise en œuvre de la pluridisciplinarité dans les services de santé au travail

The estimated domestic usage of DD (a typical nematocide) has been the greatest among all the agricultural chemicals in Japan. DD is involved in a document which is used to establish tap water quality standard in Japan. The document indicates that DD is potentially detectable in raw water for tap waters. DD in raw water will be treated with chlorine at waterworks, which raises concerns about formation of mutagenic chlorination by-products through the treatment. The objective of this work was to identify a mutagenic chlorination by-product by using high resolution mass spectrometers and multivariate analyses. Mutagenicity of the chlorination by-product was evaluated with the Ames Salmonella mutagenicity assay. (E)-1,3-Dichloropropene (DCP) was used as a model compound of DD. The mutagenicity of chlorine treated model aqueous solutions of DD increased with increasing chlorine dosages up to 3.00 mol-Cl2/mol-DCP. From the chlorine treated aqueous solutions of DD, 1,3-dichloroacetone (DCA) was identified as a major mutagen by a cochromatography with an authentic standard, precursor ion analyses and first-generation product ion analyses with the high resolution mass spectrometers. The mutagenicity of DCA against TA100 strain without microsomal activation was 160,000 net revertant colony/μmol (the toxicity equivalent factor was 0.14). Based on the mutagenicity of the chlorine treated DCP sample, the specific mutagenicity of DCA, and the DCA concentration in the sample, the mutagenic contribution of DCA to the chlorine treated DCP sample was calculated. High contribution (98%) clearly shows that DCA is a major mutagen in the chlorine treated DCP sample. Because the exploration of DCA was performed by using the unrealistic high concentration samples of DCP and chlorine, formation of DCA in the practical concentration samples (0-200 μg-DCP/L, 0.10 or 1.27 mg-Cl2/L) was examined. It was proven that DCA formation reaction takes place in the practical concentrations of DCA and chlorine.

Genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2 H-benzotriazole (PBTA-6) and 4-amino-3,3′-dichloro-5,4′-dinitro-biphenyl (ADDB) in goldfish ( Carassius auratus) using the micronucleus test and the comet assay

2-[2-(Acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2 H-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 96 h after i.p. injection of PBTA-6 and ADDB at doses of 50 mg/kg body weight, and then decreased 144 h after injection. PBTA-6 induced micronuclei in gill cells dose-dependently at a dose range of 1–100 mg/kg body weight, giving significantly high frequencies at doses of 50 and 100 mg/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 6 h after the i.p. injection of PBTA-6 (50 mg/kg body weight), only to decrease by 9 h 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 50 mg/kg. Significantly high values for tail moment and tail length were found in peripheral erythrocytes 3 h after an i.p. injection of ADDB and persisted for up to 6 h. These results show that both PBTA-6 and ADDB have genotoxic effects in goldfish.

Mutagenic activity and quantification of nitroarenes in surface soil in the Kinki region of Japan

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 10–2092 pg/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 144–1158 pg/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.