Structure-mutagenicity relationships of four amino-imidazonaphthyridines and imidazoquinolines

We tested four isomeric imidazonaphthyridines and one imidazoquinoline compound for mutagenic activity in the Ames/Salmonella mutagenicity assay, using strain TA98 and strain YG1024, an analogue of strain TA98 with elevated O-acetyl-transferase levels. Their potency was related to calculated electronic parameters. Five compounds with a linear arrangement of 3 rings showed a positive response in strain YG1024. Compound 2 (1-methyl-imidazo[4,5-b][1,7]naphthyridin-2-amine) is the most mutagenic in both strains, giving specific activities of about 200 and 30 revertants per microgram in strains YG1024 and TA98, respectively. Three of the compounds were weak mutagens, giving a positive dose-response only in strain YG1024, with 3–5 revertants per microgram. A higher response of all five compounds in strain YG1024 as opposed to TA98 indicates that they require O-acetyltransferase activity for their metabolism. Mutagenic potencies in strain YG1024 were positively correlated to the energy of the LUMO (lowest unoccupied molecular orbital) of the nitrenium ion. © 1995 Wiley-Liss, Inc.     

Characterization of mutagenic activity in instant hot beverage powders

Extracts of several grain-based coffee-substitute blends and instant coffees were mutagenic in the Ames/Salmonella test using TA98, YG1024, and YG1029 with metabolic activation. The beverage powders induced 150 to 500 TA98 and 1,150 to 4,050 YG1024 revertant colonies/g, respectively. Increased sensitivity was achieved using strain YG1024. No mutagenic activity was found in instant hot cocoa products. The mutagenic activity in the beverage powders was shown to be stable to heat and the products varied in resistance to acid nitrite treatment. Differential bacterial strain specificity, and a requirement for metabolic activation suggest that aromatic amines are present. Characterization of the mutagenic activity, using HPLC and the Ames test of the collected fractions, showed the coffee-substitute blends and instant coffees contain several mutagenic compounds. Known heterocyclic amines are not responsible for the major part of the mutagenic activity. The main mutagenic activity in grain-based coffee-substitute blends and instant coffees is due to several unidentified compounds, which are most likely aromatic amines. © 1995 Wiley-Liss, Inc.     

Evaluation of the mutagenicity of nitration products derived from phenalenone (1H-phenalen-1-one)

1H-Phenalen-1-one (phenalenone) is one of the major oxygenated polyaromatic compounds present in the atmospheric environment. In order to gain detailed information regarding the mutagenicity and physicochemical properties of the nitration products of phenalenone, we measured Ames Salmonella mutagenicity, lower LUMO (lowest unoccupied molecular orbital) energy and octanol-water partition coefficient of the products obtained from the nitration reaction of phenalenone. Both nitration reactions of phenalenone, i.e. with mixed inorganic acids (a mixture of nitric acid and sulphuric acid) and with NO(2)-O(3) in an aprotic solvent, preferentially afforded the nitration products 2-nitrophenalenone and 5-nitrophenalenone. Formation of a 6-nitro derivative of phenalenone was, however, only observed in the nitration reaction with sulphuric acid. Moreover, dinitro derivatives of phenalenone and also two oxidatively decomposed products of nitrophenalenone, i.e. 3-nitro- and 4-nitronaphthalic anhydride, were isolated from the reaction mixture. The mutagenicities of the six nitro compounds obtained from the nitration reactions were tested with the Salmonella strains TA98, TA100, YG1021 and YG1024 in the absence of S9 mix. Among these products, 2-nitrophenalenone exhibited the most potent mutagenic activity against TA98, TA100 and YG1024 (160, 230 and 2800 revertants/nmol for strains TA100, TA98 and YG1024, respectively), whereas 2,5-dinitrophenalenone exerted the highest mutagenicity against YG1021. Semi-empirical calculation showed that among the mononitrophenalenone series, the mononitro derivatives possessing lower LUMO energy tended to exhibit greater mutagenic activity than those with higher LUMO energy. This tendency, however, did not extend to the compounds with different aromatic ring systems due to the considerable differences in the hydrophobicities of these compounds.     

Urinary mutagenicity on TA98 and YG1024 Salmonella typhimurium strains after a hamburger meal: influence of GSTM1 and NAT2 genotypes

Mutagenicity on TA98 and YG1024 Salmonella typhimurium strainsof pan–fried hamburger extracts and of 24 h post–mealurine from 32 non–smoking volunteers was evaluated. Eachparticipant in the study was GSTM1 and NAT2 genotyped. Aftercooking the meat showed mutagenic activity (mean ± SD)on strains TA98 and YG1024 of 114 ± 129 and 1437 ±1536 net revertants/g respectively. Twenty three of 32 urinesamples showed clear mutagenic activity (i.e. caused at leasta doubling of the number of spontaneous revertants) on the 0-acetyltransferaseoverproducing strain YG1024, while none of the post-meal 24h urine samples was clearly mutagenic on strain TA98. Total24 h post–meal YG1024–active urinary mutagens werewell correlated with the levels of mutagen intake with the meal(r² = 0.5977, F = 44.58, P < 0.01). In the group under studyGSTM1 genotypes did not influence urinary mutagenicity. Highlyexposed subjects (n = 15) with the NAT2–ss genotype showedsignificantly increased levels of urinary mutagenicity on strainYG1024 in comparison with NAT2-R subjects (mutagen intake-adjustedtotal 24 h mutagen excretion = 1.00 ± 0.29 versus 0.66± 0.32, Mann-Whitney U test, U = 12.5, P < 0.05).Our results suggest that the levels of urinary mutagens derivedfrom diets rich in heterocyclic aromatic amines, which are specificallydetected by the YG1024 Salmonella strain, are modulated by NAT2-dependentenzyme activity, slow acetylators having higher levels of mutagensin their urine. Subjects with the rapid acetylator genotype,who are known to be at risk for colon cancer, seem to be partiallyprotected with respect to the risk of bladder cancer. ⁴To whom correspondence should be addressed. Tel: 498216637; Fax: 498216621; Email: clonfero{at}uxl.unipd.it     

Identification of mutagens in freshwater sediments by the Ames-fluctuation assay using nitroreductase and acetyltransferase overproducing test strains

Extracts of sediments from an area of concern in the Elbe river basins (Spittelwasser creek) were analyzed with the Ames-fluctuation test and in parallel with gas chromatography/mass spectrometry for compound identification. The standard test strains TA 98 and TA 100 showed mutagenicity mainly in medium-polar fractions of the sediment extracts. PAHs contribute to the overall mutagenic potential of the sample. Especially, cyclopenta[c,d]pyrene that was previously not defined as a priority hazardous substance has to be considered as well. The addition of metabolically competent test strains, which overexpress nitroreductase and acetyltransferase (e.g., YG1041 and YG1042) to the test battery, increased significantly the sensitivity of the Ames test for medium polar to polar genotoxins. The increased mutagenicity that was found in these bacterial strains indicates the presence of nitroarenes and/or aromatic amines. In fact, a number of heterocyclic and nitrogen-substituted aromatic compounds were identified in the sediments of the Spittelwasser creek of which methyl parathion, 1-naphthylamine, and N-phenyl-2-naphthylamine are mutagenic.     

Unicellular green alga Chlamydomonas reinhardtii as an activation system for 2-aminofluorene

Despite the promutagenic/procarcinogenic potential, polycyclic aromatic amines are widely spread in the environment. Biotransformation of the polycyclic aromatic amine 2-aminofluorene (2-AF) was proved in mammals and higher plants. The algal cell/microbe coincubation assay is an additional system that complemented those proved in mammals and higher plants, useful for detection and conversion of environmental promutagens, mainly in aquatic environments. The unicellular green algae may be a good activating system in coincubation assays in that the algal cells exist as a natural system. To increase the effectiveness of this metabolizing system, different modifications of the standard experimental procedure were conducted. Algae can accumulate and metabolize promutagenic pollutants, some of which may differ from those activated by the animal microsome metabolizing system (S9 mix) and by the plant cell/microbe coincubation assay. 2-AF was activated in the algal cell/microbe coincubation assay in which wild-type Chlamydomonas reinhardtii cells were used as an activating system and the bacteria Salmonella typhimurium TA98, YG1024, and yeast Saccharomyces cerevisiae D7 as the genetic indicator organisms. It was converted to the mutagenic product(s) for the strain YG1024, but the strain TA98 did not exhibit any increase in the mutant yield of His⁺ revertants. Consequently, metabolites from 2-AF are substrates for O-acetyltransferase. A direct comparison of algal 2-AF activation with mammalian activation system (S9 mix) proved the higher activity of mammalian microsome system (S9 mix). After the combination of both activation systems, a slight synergetic effect was found. Although the genetic endpoints induced by 2-AF using both modifications of the algal cell/S. cerevisiae coincubation assay and those obtained in intact yeast cells were similar at the equitoxic concentrations, 2-AF activation by the algal supernatant slightly increased the genetic endpoints studied. Environ. Mol. Mutagen. 31:383–389, 1998 © 1998 Wiley-Liss, Inc.     

Combining different assays and chemical analysis to characterize the genotoxicity of waters impacted by textile discharges

Waters receiving textile discharges can exhibit genotoxic and mutagenic activity, which has been related to the presence of dyes and aromatic amines as synthesis precursors or byproducts. The aim of this study was to identify dyes and aromatic amines in water samples impacted by textile discharges, and to evaluate the genotoxic responses of these samples using the Salmonella/microsome assay in strains TA98 and YG1041, and the Fpg‐modified comet assay in the RTL‐W1 fish cell line. The genotoxicity of river samples downstream of the discharge was greater than the upstream samples in both of the Ames tests. The Fpg‐modified comet assay detected similar levels of DNA damage in the upstream and downstream samples. Mutagenicity was not detected with TA98, except for the Quilombo River samples, but when YG1041 was used as the tester strain mutagenicity was detected for all sites with a very different profile in upstream sites relative to the other sites. The mutagenic response strongly indicated that aromatic amines or dyes were contributing to the mutagenic activity downstream. The impact of textile discharges was also confirmed by chemical analysis, because the highest concentrations of azo dyes and aromatic amines were detected in the river downstream. This study shows the value of combining assays measuring complementary endpoints to better characterize the mutagenicity of environmental samples, with the advantage that this approach provides an indication of what classes of compounds are responsible for the effect. Environ. Mol. Mutagen. 57:559–571, 2016. © 2016 Wiley Periodicals, Inc.     

Reduction in the mutagenicity of synthetic dyes by successive treatment with activated sludge and the ligninolytic fungus, Irpex lacteus

Synthetic dyes are released in wastewater from textile manufacturing plants, and many of these dyes are genotoxic. In the present study, the mutagenicity of azo, anthraquinone, and triphenyl methane dyes was investigated before and after successive biodegradation with activated sludge and the ligninolytic fungus, Irpex lacteus. Two biodegradation systems were used to reduce the genotoxicity of dyes that were not efficiently inactivated by activated sludge alone. Mutagenicity was monitored with the Salmonella reversion assay conducted with the base-pair substitution detector strains, TA100 and YG1042, and the frame-shift detector strains, TA98 and YG1041, with and without rat liver S9. All dyes except for Congo Red (CR) were mutagenic with S9 activation. Assays conducted with the dyes indicated that only the azo dye Reactive Orange 16 (RO16) was mutagenic in both TA98 and TA100. Methyl Red and Disperse Blue 3 (DB3) were mutagenic in TA98, YG1041 and YG1042, while Reactive Black 5 was mutagenic in YG1041 and YG1042. Remazol Brilliant Blue R (RBBR), Crystal violet (CV) and Bromophenol Blue (BPB) were mutagenic only in TA98, but the toxicity of the latter two dyes complicated the evaluation of their mutagenicity. CR was not mutagenic in any of the tester strains. Biodegradation studies conducted with RO16 and DB3 indicated that the two-step biodegradation process reduced the mutagenic potential of RO16 and DB3 to a greater extent than activated sludge alone; the mutagenicity of the two dyes was reduced by 95.2% and 77.8%, respectively, by the two-step process. These data indicate that the combined biodegradation process may be useful for reducing the mutagenicity associated with wastewater from textile factories that contain recalcitrant dyes.     

Comparisons of mutation induction by six monocyclic aromatic amines in Salmonella typhimurium tester strains TA97, TA1537, and TA1538

The mutagenicity of six monocyclic aromatic amines (2,4-diaminoanisole, 2,4-diaminoethoxybenzene, 2,4-diaminopropoxybenzene, m-phenylenediamine, 2,4-diaminotoluene, and nitro-p-phenylenediamine) was investigated in Salmonella typhimurium strains TA97, TA1537, and TA1538 in the presence of two different amounts of Aroclor 1254-induced S9 preparations. Strain TA1538 was found to be the most responsive of the three strains with this group of compounds. Regarding the other strains, TA1537 responded to three of the compounds better than strain TA97, if one calculates responsiveness as the fold-increase in numbers of revertants per plate. However, if one calculates the number of revertants per nanomole or compares the number of induced revertants per plate, TA97 was more responsive than TA1537 for all six compounds. Comparisons of mutagenesis from tests involving strain TA97 are complicated by the large variations in spontaneous mutation frequencies in this strain. The amount of S9 per plate is another important variable in tests of monocyclic aromatic amines; in general, more revertants are detected when the S9 mix contains 10% S9 than when it contains 4% S9. Nevertheless, in all our tests of 2,4-diaminoanisole, 2,4-diaminoethoxybenzene, and 2,4-diaminopropoxybenzene, the same relationship between chemical structure and mutagenic activity was observed. In all three strains, the mutagenic responses become less when the alkoxy substituent on the molecule becomes larger.     

Plant-activation of the bicyclic aromatic amines benzidine and 4-aminobiphenyl

Benzidine and 4-aminobiphenyl (4-ABP) are promutagenic bicyclic aromatic amines that are activated into frameshift and base pair substitution mutagens by plant systems. Using the plant cell/microbe coincubation assay, plant-activated benzidine from 0 to 50 μM induced a concentration-response in Salmonella typhimurium. At concentrations above 5 μM, plant-activated benzidine induced frameshift and base pair substitution mutations in the N- or O-acetyltransferase over-expressing strains, DJ460, YG1024, and YG1029. With plant-activated 4-ABP, concentrations above 250 μM induced a significant mutagenic response in strains YG1024 and YG1029. A tobacco cell-free mixture, TX1MX, activated benzidine and 4-ABP into mutagenic metabolites in S. typhimurium strains YG1024, YG1029, and DJ460. The mutagenic sensitivities of plant-activated benzidine and 4-ABP were the same with two different types of plant activation systems, TX1 suspension cells and TX1MX cell-free medium. The plant activation of these aromatic amines is mediated by tobacco cell peroxidase. Plant-activated benzidine and 4-ABP are converted into intermediates that serve as substrates for bacterial or humanacetylCoA: N-hydroxyarylamine N-acetyl-transferase to generate the ultimate mutagenic products. Environ. Mol. Mutagen. 29:81–90, 1997 © 1997 Wiley-Liss, Inc.     

Mutagenicity of an aged gasworks soil during bioslurry treatment

This study investigated changes in the mutagenic activity of organic fractions from soil contaminated with polycyclic aromatic hydrocarbons (PAHs) during pilot‐scale bioslurry remediation. Slurry samples were previously analyzed for changes in PAH and polycyclic aromatic compound content, and this study examined the correspondence between the chemical and toxicological metrics. Nonpolar neutral and semipolar aromatic fractions of samples obtained on days 0, 3, 7, 24, and 29 of treatment were assayed for mutagenicity using the Salmonella mutation assay. Most samples elicited a significant positive response on Salmonella strains TA98, YG1041, and YG1042 with and without S9 metabolic activation; however, TA100 failed to detect mutagenicity in any sample. Changes in the mutagenic activity of the fractions across treatment time and metabolic activation conditions suggests a pattern of formation and transformation of mutagenic compounds that may include a wide range of PAH derivatives such as aromatic amines, oxygenated PAHs, and S‐heterocyclic compounds. The prior chemical analyses documented the formation of oxygenated PAHs during the treatment (e.g., 4‐oxapyrene‐5‐one), and the mutagenicity analyses showed high corresponding activity in the semipolar fraction with and without metabolic activation. However, it could not be verified that these specific compounds were the underlying cause of the observed changes in mutagenic activity. The results highlight the need for concurrent chemical and toxicological profiling of contaminated sites undergoing remediation to ensure elimination of priority contaminants as well as a reduction in toxicological hazard. Moreover, the results imply that remediation efficacy and utility be evaluated using both chemical and toxicological metrics. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Mechanistic QSAR of aromatic amines: new models for discriminating between homocyclic mutagens and nonmutagens, and validation of models for carcinogens

Because of its environmental and industrial importance, the aromatic amines are the single chemical class most studied for its ability to induce mutations and cancer. The large database of mutagenicity and carcinogenicity results has been studied with Quantitative Structure-Activity Relationship (QSAR) approaches by several authors, leading to models for the following: (a) the mutagenic potency in Salmonella thyphimurium; (b) the carcinogenic potency in rodents; and (c) the discrimination between rodent carcinogens and noncarcinogens. However, satisfactory models for the discrimination between mutagens and nonmutagens are lacking. The present work provides new QSARs for mutagenic/nonmutagenic homocyclic aromatic amines in S. typhimurium strains TA98 and TA100. The two new models are validated by checking their ability to predict the mutagenicity of further aromatic amines not included in the training set, and not used to generate the QSAR models. In addition, we also validated previous QSAR models for the carcinogenicity/noncarcinogenicity of the aromatic amines with external data. The mechanistic implications of the models are discussed in light of the other QSARs for the aromatic amines. The results of the analysis point to two QSAR models (one for mutagenicity and one for rodent carcinogenicity) as reliable tools for the in silico characterization of the risk posed by the aromatic amines.     

Detection of direct-acting mutagens in ambient air: a comparison of two highly sensitive mutagenicity assays.

Ambient air has been shown to contain numerous hazardous pollutants, many of which are known or suspected carcinogens and mutagens. Bioassays play a prominent role in the characterization of these genotoxic pollutants, and as new test methods are developed, it is incumbent upon researchers to evaluate assay performance and report relative merits. In this study, two Salmonella test methods (the spiral and preincubation assays) were assessed to determine their usefulness as screening methods for monitoring direct-acting mutagens in ambient air. The spiral assay automates the conventional plate-incorporation assay and has been shown to reduce the labor, materials, and sample mass required to perform mutagenicity testing. The preincubation assay has been shown to enhance test sensitivity for certain classes of compound, thereby reducing the amount of sample required for dose-response analysis. Both assays were used to test organic extracts of airborne particulate matter collected in Tokyo during the winters of 1988 and 1990. In addition to the conventional tester strains TA98 and TA100, two newly developed YG strains were evaluated. Strains YG1024 and YG1029-derived from TA98 and TA100, respectively-contain an acetyltransferase plasmid that confers upon the strains greater sensitivity towards nitroarenes. Results from this study indicated that both assays were able to detect direct-acting mutagens in the Tokyo air samples. The mutagenic activity associated with the samples was directly related to the particle mass present in a given volume of air. Mutagenic response was greater in the spiral assay relative to the preincubation assay, especially when YG tester strains were used. The YG strains were significantly more sensitive to mutation than the TA strains in both assays, which suggests that nitroaromatics are an important class of genotoxic contaminant present in Tokyo air.     

A promising Ames battery for mutagenicity characterization of new dyes

When testing new products, potential new products, or their impurities for genotoxicity in the Ames test, the quantity available for testing can be a limiting factor. This is the case for a dye repository of around 98,000 substances the Max Weaver Dye Library (MWDL). Mutagenicity data on dyes in the literature, although vast, in several cases is not reliable, compromising the performance of the in silico models. In this report, we propose a strategy for the generation of high‐quality mutagenicity data for dyes using a minimum amount of sample. We evaluated 15 dyes from different chemical classes selected from 150 representative dyes of the MWDL. The purity and molecular confirmation of each dye were determined, and the microplate agar protocol (MPA) was used. Dyes were tested at the limit of solubility in single and concentration‐response experiments using seven strains without and with metabolic activation except for anthraquinone dyes which were tested with eight strains. Six dyes were mutagenic. The most sensitive was YG1041, followed by TA97a > TA98 > TA100 = TA1538 > TA102. YG7108 as well as TA1537 did not detect any mutagenic response. We concluded that the MPA was successful in identifying the mutagenicity of dyes using less than 12.5 mg of sample. We propose that dyes should be tested in a tiered approach using YG1041 followed by TA97a, TA98, and TA100 in concentration‐response experiments. This work provides additional information on the dye mutagenicity database available in the literature.     

Mutagenicity of blue rayon extracts of fish bile as a biomarker in a field study

Blue rayon (BR) in combination with the Salmonella/microsome assay was used to evaluate the mutagenicity of fish bile samples. Specimens of Mugil curema from two sites were collected over a 1‐year period. Piaçaguera channel contains high concentrations of total polycyclic aromatic hydrocarbons (PAHs) and other contaminants, while Bertioga channel was considered the reference sites in this study. Bile was extracted with BR and tested with TA98, TA100, and YG1041 strains with and without S9 in dose response experiments. PAH metabolite equivalents were analyzed using reverse‐phase high performance liquid chromatography /fluorescence. Higher mutagenic responses were observed for the contaminated site; YG1041 with S9 was the most sensitive strain/condition. Mutagenicity ranged from 3,900 to 14,000 rev./mg at the contaminated site and from 1,200 to 2,500 rev./mg of BR at the reference site. The responses of YG1041 were much higher in comparison with the TA98 indicating the presence of polycyclic compounds from the aromatic amine class that cause frameshift mutation. TA100 showed a positive mutagenic response that was enhanced following S9 treatment at both sites suggesting the presence of polycyclic compounds that require metabolic activation. benzo(a)pyrene, naphthalene, and phenanthrene metabolite equivalents were also higher in the bile of fish collected at the contaminated site. It was not possible to correlate the PAH metabolite quantities with the mutagenic potency. Thus, a combination of the Salmonella/microsome assay with YG1041 with S9 from BR bile extract seems to be an acceptable biomarker for monitoring the exposure of fish to mutagenic polycyclic compounds. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

1-nitropyrene as a marker for the mutagenicity of diesel exhaust-derived particulate matter in workplace atmospheres

The use of 1-nitropyrene (1-NP) as a marker for the occupational exposure to diesel exhaust (DE) mutagens was investigated in workplace atmospheres contaminated with DE from a variety of emission sources, such as power supplies, forklifts, trucks, caterpillar vehicles, trains, ships' engines, and vehicles in city traffic. Total suspended particulate matter was collected by area sampling. The 1-NP content of acetone extracts of these samples as determined by gas chromatography-high resolution mass spectrometry varied from 0.080 to 17 μg/g acetone extractable matter, corresponding to air concentrations of 0.012 to 1.2 ng/m³. A sample collected in a rural area contained 0.0017 ng/m³ 1-NP. The mutagenicity of the extracts was tested in the Salmonella typhimurium strains TA98 and TA1538, using the microsuspension assay with and without metabolic activation by an exogeneous metabolizing system (rat liver S9-fraction). In addition, the S. typhimurium strains YG1021 and YG1024 were used because of their high sensitivity towards the mutagenicity of nitro polycyclic aromatic hydrocarbons. When plotting the mutagenic potency of the air sample extracts as determined in the absence of liver S9 versus the particle-associated 1-NP level, a relatively high correlation (r = 0.80–0.91) was observed in all of the S. typhimurium strains. High correlations (r = 0.80–0.93) were also observed when plotting the results of mutagenicity testing after activation by S9 versus the outcome of chemical analysis. These results show that the 1-NP content of workplace air samples is associated with their mutagenic potency, suggesting that 1-NP may be used as a marker for occupational exposure to DE-de-rived particle-associated mutagens © 1995 Wiley-Liss, Inc.     

Mutagenic activities and physicochemical properties of selected nitrobenzanthrones

Mutagenic activity of nine nitro derivatives of benzanthrone, namely 1-nitro-, 2-nitro-, 3-nitro-, 9-nitro-, 11-nitro-, 1,9-dinitro-, 3,9-dinitro-, 3,11-dinitro- and 3,9,11-trinitrobenzanthrone were tested with Salmonella strains TA98, TA100, YG1021 and YG1024 in both the presence and absence of an S9 mix. Each compound exhibited mutagenic activity with all the strains. Among these nine isomers, 3-nitrobenzantrone exhibited the most mutagenic activity with all the strains without the S9 mix. The mutagenic activities of the dinitro and trinitro derivatives of benzanthrone were lower than that of the 3-nitro derivative; this is evident from the mutagenic activity of nitrated polyaromatic hydrocarbons (PAH), which is generally enhanced with an increase in nitration. The physicochemical properties of nitrated benzanthrone (reduction potential, hydrophobicity and orientation of nitro groups to the aromatic ring) demonstrated that mononitrated benzanthrone exhibits a lower reduction potential than mononitroPAHs such as 1-nitropyrene and 3-nitrofluoranthene, but was almost equivalent to that of dinitroPAH. Moreover, the mutagenic activity of mononitrobenzanthrones clearly depend on the reduction potential of each compound; however, this tendency was not observed in polynitrobenzanthrones, probably because the reduction of the nitro groups to amino groups of polynitrated benzanthrone might be predominant without a sufficient formation of corresponding hydroxyamines. These results suggest that aromatic compounds that contain keto groups, when nitrated, may act as potentially powerful direct-acting mutagens.     

Mutagenic activation of 4-aminobiphenyl and its N-hydroxy derivatives by microsomes from cultured human uroepithelial cells

Activation of the human bladder carcinogen 4-aminobiphenyl (ABP)and its N-hydroxy derivatives was investigated using lysatesand subcellular enzyme preparations from cultured human uroepithelialcells (HUC). Mutagenic activation was determined using Salmonellatyphimurium strains TA98; TA98/1,8-DNP₆, a derivative deficientin acetyl coenzyme A:N-hydroxyarylamine O-acetyltransferase(OAT); and YG1024, a derivative of TA98 with elevated OAT activityand enhanced sensitivity to mutation by N-hydroxyarylamines.Mutagenicity of ABP catalyzed by HUC microsomes was detectedin YG1024 but not hi the parent strain TA98. HUC microsomesalso catalyzed the mutagenic activation of N-hydroxy-4-acetylaminobiphenyl(N-OH-AABP) and the relative sensitivity of the tester strainswas YG1024 > TA98 > TA98/1,8-DNP₆, indicating N-hydroxy-4-aminobiphenyl(N-OH-ABP) as the mutagenic intermediate. In contrast, the mutagenicactivity of N-acetoxy-4-acetylaminobiphenyl incubated with HUCmicrosomes was approximately equal in TA98 and YG1024, and mayinvolve N-acetoxy-4-aminobiphenyl (N-OAc-ABP) as the intermediate.High pressure liquid chromatography (HPLC) of the DNA hydrolysateobtained after incubation of [³H]N-OH-ABP with YG1024, showedN-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-ABP) as the primaryadduct, based on mobility of the radioactivity in comparisonwith the synthetic standard. Additionally, HUC microsomes catalyzedthe binding of [³H]N-OH-ABP to RNA in the presence of 4-acetylaminobiphenyl(AABP), N-OH-AABP and acetyl coenzyme A as acetyl donors, andthis binding was blocked by paraoxon. The hydrolysate obtainedfrom incubation of DNA with [³H]N-OH-ABP and HUC microsomes,with AABP as acetyl donor, revealed the formation of dG-ABPadduct. ³²P-post-labeling of adducted DNA from N-OH-ABP, N-OH-AABPand N-OAc-AABP showed similar adduct profiles, suggesting thatthe aryl nitrenium ion, arising from N-OAc-ABP, might be thereactive species responsible for the mutagenic activity. ¹To whom correspondence should be addressed     

Evaluation of the genotoxicity of river sediments from industrialized and unaffected areas using a battery of short-term bioassays

The present investigation evaluated the capacity of the Salmonella mutagenicity test, the comet assay, and the micronucleus assay to detect and characterize the genotoxic profile of river sediments. Three stations were selected on an urban river (Bouches du Rhône, France) exposed to various sources of industrial and urban pollution (StA, StB, and StC) and one station on its tributary (StD). One station in a nonurban river was included (REF). The concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) were determined by HPLC, and the genotoxicity of the sediments was monitored by the Salmonella mutagenicity test (TA98 + S9, YG1041 ± S9), the comet assay, and the micronucleus assay on CHO cells. Chemical analysis showed that the total PAH concentrations ranged from 23 μg kg^?1 dw (REF) to 1285 μg kg^?1 dw (StD). All the sediments were mutagenic in the Salmonella mutagenicity test. The mutagenicity was probably induced by the presence of nitroarenes (StA, StB, StC, and StD) and aromatic amines (REF) as deduced from the mutagenicity profiles of strains YG1041 ± S9 and TA98 + S9. The comet assay revealed direct DNA lesions in REF, StA, and StB sediments and metabolization‐dependent DNA damage in StC and StD. The micronucleus assay showed an absence of clastogenicity for StA ± S9 and StC‐S9, and a significant clastogenicity ± S9 for the three other stations. The genotoxicity ranking determined by the comet assay + S9 matched the ranking of total and carcinogenic PAH concentrations, and this assay was found to be the most sensitive. Environ. Mol. Mutagen., 2008. © 2008 Wiley‐Liss, Inc.     

Genotoxicity of nitrosulfonic acids, nitrobenzoic acids, and nitrobenzylalcohols, pollutants commonly found in ground water near ammunition facilities

2-Amino-4,6-dinitrobenzoic acid (2-A-4,6-DNBA), 4-amino-2,6-dinitrobenzoic acid (4-A-2,6-DNBA), 2,4,6-trinitrobenzoic acid (2,4,6-TNBA), 2-amino-4, 6-dinitrobenzylalcohol (2-A-4,6-DNBAlc), 4-amino-2,6-dinitrobenzylalcohol (4-A-2,6-DNBAlc), 2,4-dinitrotoluol-5-sulfonic acid (2,4-DNT-5-SA), 2,4-dinitrotoluol-3-sulfonic acid (2,4-DNT-3-SA), and 2, 4-dinitrobenzoic acid (2,4-DNBA) are derivatives of nitro-explosives that have been detected in groundwater close to munitions facilities. In the present study, the genotoxicity of these compounds was evaluated in Salmonella/microsome assays (in strains TA100 and TA98, with and without S9 and in TA98NR without S9), in chromosomal aberration (CA) tests with Chinese hamster fibroblasts (V79), and in micronucleus (MN) assays with human hepatoma (HepG2) cells. All compounds except the sulfonic acids were positive in the bacterial mutagenicity tests, with 2,4,6-TNBA producing the strongest response (8023 revertants/micromol in TA98 without S9 activation). 2-A-4,6-DNBA was a direct acting mutagen in TA98, but negative in TA100. The other positive compounds were approximately 1-3 orders of magnitude less mutagenic than 2,4,6-TNBA in TA98 and in TA100; relatively strong effects ( approximately 50-400 revertants/micromol) were produced by the benzylacohols in the two indicator strains. With the exception of 2,4-DNBA, the mutagenic responses were lower in the nitroreductase-deficient strain TA98NR than in the parental strain. 2,4-DNBA produced a marginally positive response in the V79-cell CA assay; the other substances were devoid of activity. Only the benzoic acids were tested for MN induction in HepG2 cells, and all produced positive responses. As in the bacterial assays, the strongest effect was seen with 2,4,6-TNBA (significant induction at >or=1.9 microM). 4-A-2,6-DNBA was positive at 432 microM; the weakest effect was observed with 2,4,-DNBA (positive at >or=920 microM). The differences in the sensitivity of the indicator cells to these agents can be explained by differences in the activities of enzymes involved in the activation of the compounds. The strong responses produced by some of the compounds in the human-derived cells suggest that environmental exposure to these breakdown products of nitro-explosives may pose a cancer risk in man.