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.     

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.     

Prostaglandin H synthase-dependent genotoxicity of 2,4-diaminotoluene.

2,4-Diaminotoluene (2,4-DAT), a high volume synthetic compound, is moderately carcinogenic to rodents. We report here that 2,4-DAT is a substrate for the peroxidase activity of prostaglandin H synthase (PHS). In contrast to many aromatic amines which are activated as mutagens by PHS, we find that 2,4-DAT is not mutagenic to six S. typhimurium strains with this activation system. The strains tested include YG1006, YG1024, and YG1029, which are far more sensitive to the mutagenicity of aromatic amines and nitroarenes than are the standard tester strains. Although not mutagenic itself, 2,4-DAT does enhance the mutagenicity of 2-aminofluorene (2-AF) in the PHS-catalyzed system in strains TA98, YG1006, and YG1024, with maximal enhancement of 140%, 1831%, and 1216%, respectively. Half-maximal enhancement of 2-AF mutagenicity is observed at 15-20 microM 2,4-DAT for strains YG1006 and YG1024, and about 80 microM for TA98. Studies with compounds structurally related to 2,4-DAT revealed enhancement of 2-AF mutagenicity with 2,5-DAT and o-phenylenediamine (o-PD) but not for other DAT isomers, toluidines, and phenylenediamines. Maximal enhancement of 2-AF mutagenicity observed in TA98 with PHS-catalyzed activation was 110% for o-PD and 60% for 2,5-DAT. This comutagenic effect of 2,4-DAT appears quite specific for 2-AF, as it fails to enhance either the PHS-dependent mutagenicity of the aromatic amines benzidine and 2-naphtylamine, or the direct mutagenicity of N-acetoxy-acetylaminofluorene,2-nitrofluorene,4- nitroquinoline-N-oxide and 1,1,1-trichloropropene-2,3-oxide. Enhancement of 2-AF mutagenicity by 2,4-DAT is also observed with cytochrome P-450-dependent activation, however the half-maximal 2,4-DAT concentration was 400 microM, and the maximal enhancement was only 50%. The ability of 2,4-DAT, under conditions where it is not itself mutagenic, to enhance the genotoxicity of the potent carcinogen 2-AF comprises an intriguing toxicological interaction, and underscores the inherent difficulties in assessing the genotoxic risks posed by mixtures of compounds.     

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.     

Comparative mutagenicity of nine brands of coffee to Salmonella typhimurium TA100, TA102, and TA104

Nine coffee preparations, four caffeinated instant brands, three caffeinated drip coffees, and two decaffeinated coffees, one of which was an instant brand, were evaluated for mutagenicity by the Ames assay using Salmonella typhimurium TA100, TA102, and TA104. All the coffees contained direct-acting mutagens, which reverted the three strains. The inclusion of a rat microsomal enzyme preparation reduced the mutagenic response of the three strains in the presence of some of the coffee samples. Both glyoxal and methylglyoxal, 1,2-dicarbonyls found in the coffees were mutagenic. The concentration of glyoxal, methyglyoxal, diacetyl, and guiacol were measured by gas chromatography/mass spectrometry. Caffeine, furfural, and 5-methylfurfural concentrations were determined by high performance liquid chromatography. Although lower concentrations of methyglyoxal were found in the drip caffeinated coffees, the mutagenic potency of these preparations was higher than the instant coffees on a weight basis especially when TA104 was the indicator organism. Our findings agree with those of other workers who have shown that carbonyl compounds, which were present in all the brands tested, are partially responsible for the mutagenic response of coffee but that additional mutagens are also present.     

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.     

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.     

Coffee is highly mutagenic in the L-arabinose resistance test in Salmonella typhimurium

The present study shows that the L-arabinose resistance test in Salmonella typhimurium detects coffee as a strong mutagen in the absence of mammalian microsomal activation. The response of the Ara forward mutation assay was 8.5 times higher than that of TA104, which is the most sensitive to coffee of the tester strains of the Ames test. Both the mutagenesis protocol (preincubation test) and the additional genetic characteristics of the bacterial tester strain (excision repair deficiency, normal lipopolysaccharide barrier, and the presence of plasmid pKM101) were critical factors in the optimal induction by coffee of forward mutations to L-arabinose resistance. All ten samples of roasted coffee analyzed with the Ara assay were highly mutagenic: one cup of coffee (150 ml) was calculated to induce 3-4 X 10(6) AraR mutants. In contrast, coffee prepared from unroasted beans (green coffee) had no mutagenic activity. Regular- and sugar-roasted coffees showed similar mutagenicities, but the specific mutagenic activity of instant coffees (1559 AraR mutants/mg) was almost 2 times that of noninstant ones (834 AraR mutants/mg). The Ara assay allowed the direct testing of coffee, although it was demonstrated that lyophilization has no effect on the mutagenicity of this beverage. Like roasted coffee, roasted barley induced a large number of AraR mutants per mg (227), though its specific mutagenic activity was approximately 4 and 7 times lower than that of noninstant and instant coffees, respectively.     

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.     

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.     

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     

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.     

A QSAR investigation of the role of hydrophobicity in regulating mutagenicity in the ames test: 1. Mutagenicity of aromatic and heteroaromatic amines in Salmonella typhimurium TA98 and TA100

Quantitative structure-activity relationships (QSAR) have been derived for the mutagenic activity of 88 aromatic and heteroaromatic amines acting on Salmonella typhimurium TA98 + S9 and 67 amines acting on TA100 + S9. Mutagenic activity is linearly dependent on hydrophobicity, the energy of the highest occupied molecular orbital, and the energy of the lowest unoccupied molecular orbital of the amine. The dependence of mutagenic activity on hydrophobicity and electronic effects is nearly identical for TA98 and TA100. Mutagenic activity in TA98 is also found to depend on the size of the aromatic ring system. Different QSARs are derived for the mutagenic activity of hydrophilic amines (log P < 1) acting on either TA98 or TA100. The mechanism of amine activation and reaction with DNA is considered in light of these findings.     

Mutagenicity of some benzidine congeners and their N-acetylated and N,N'-diacetylated derivatives in different strains of Salmonella typhimurium

The Ames Salmonella/microsome test was used to compare the mutagenic response of Salmonella typhimurium TA100, TA98, TA1538, and TA1535 to 12 benzidine derivatives, ie, benzidine, 3,3'-dimethoxybenzidine, 3,3'-dimethylbenzidine, 3,3'-dichlorobenzidine, and the corresponding N- and N,N'-diacetylated derivatives. With a few exceptions, the mutagenic response to this series of compounds varied in the order TA98 greater than TA1538 greater than TA100 greater than TA1535 = 0, and the N-monoacetylated derivatives were more mutagenic than either the parent diamines or the N,N'-diacetyl derivatives. The relative mutagenicities of the parent amines for TA98 were 3,3'-dichlorobenzidine much greater than 3,3'-dimethoxybenzidine greater than benzidine greater than 3,3'-dimethylbenzidine.     

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 profile of atmospheric particulate matter in a small urban center subjected to airborne emission from vehicle traffic and sugar cane burning

Atmospheric particulate matter (PM) is genotoxic and recently was classified as carcinogenic to humans by the International Agency for Research on Cancer. PM chemical composition varies depending on source and atmospheric conditions. The Salmonella/microsome assay is the most used mutagenicity test and can identify the major chemical classes responsible for observed mutagenicity. The objective of this work was to characterize the mutagenicity of PM samples from a countryside city, Limeira, Brazil, which is influenced by heavy traffic and sugar cane biomass burning. Six samples of total PM were collected. Air mass backward trajectories were calculated. Organic extracts were assayed using the Salmonella/microsome microsuspension mutagenicity assay using TA98, YG1041, and TA1538, with and without metabolic activation (S9). YG1041 was the most sensitive strain and mutagenicity reached 9,700 revertants per m³ without metabolic activation. Potency for TA1538 was higher than TA98, indicating that this strain should be considered in air mutagenicity studies. The increased response to YG1041 relative to TA98, and the decreased response with S9, suggests that nitroaromatics are the major contributors. Limeira is among the most mutagenic cities in the world. High mutagenicity in Limeira seems to occur when the air mass from the area of sugarcane production is mixed with air from the region impacted by anthropogenic activities such as traffic. An increase in the formation of nitro‐polycyclic aromatic hydrocarbons may result from longer contact time between the aromatic compounds and the atmosphere with high NOx and ozone concentration, although more studies are required to confirm this hypothesis. Environ. Mol. Mutagen. 57:41–50, 2016. © 2015 Wiley Periodicals, Inc.     

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.     

Microbial mutagenicity of isomeric two-, three-, and four-ring amino polycyclic aromatic hydrocarbons

The isomers of various two-, three-, and four-ring amino polycyclic aromatic hydrocarbons were tested for mutagenic activity using a microbial plate incorporation test with four Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537). All compounds were assayed with an S9 metabolic activating enzyme system. The two-ring compounds were tested only with TA98. All were weakly mutagenic (1-10 rev/micrograms) except 2-aminobiphenyl, which was not mutagenic under these test conditions. All except two of the 13 fused three-ring compounds (aminofluorenes, aminoanthracenes, and aminophenanthrenes) were active frame shift mutagens; only the aminophenanthrenes were active base-pair mutagens. The potency of this group of isomeric compounds ranged from moderately (approximately 20 rev/microgram) to strongly (greater than 5,000 rev/microgram) mutagenic. As a group, the pericondensed four-ring amino compounds were the most mutagenic of the three groups tested. All of the aminofluoranthene and aminopyrene isomers showed significant mutagenic activity with TA98, TA100, and TA1537. In general, the mutagenic potency of the amino polycyclic aromatic compounds tested was highly dependent on the structural position of the amino group.     

Salmonella/human S9 mutagenicity test: a collaborative study with 58 compounds

A large and extensive body of data on the use of human liver S9 fractions in the Salmonella mutagenicity test (Ames test) is presented; the data were obtained from a collaborative study by JEMS/BMS (Bacterial Mutagenicity Test Study Group) members and the Human and Animal Bridging Research Organization (HAB). In this study, the mutagenicity of 58 chemicals, many of which were judged to be human carcinogens by the IARC, was determined by the Ames test (the pre-incubation method at 37 degrees C for 20 min) in the presence of a selected human liver S9 fraction with a high drug-metabolic activity or a pooled human liver S9 fraction with a moderate drug-metabolic activity. For reference, mutagenicity was also examined in the presence of a phenobarbital/5,6-benzoflavone-pretreated rat liver S9 fraction, which is normally used in mutagenicity testing systems. The bacterial test strains consisted of Salmonella typhimurium TA100, TA98 or YG7108. The data indicated that the mutagenicity of chemicals in the rat and human liver S9 fractions varied considerably, depending on the chemicals in question. In addition, a large inter-individual diversity in the mutagenic response to mutagens, depending on the chemical structures of the mutagens, was also demonstrated using two selected human S9 fractions. Most of the mutagens tested in this study (75%; 36 out of 48 compounds that were judged to be mutagenic in at least one S9 fraction) were less mutagenic in the presence of the two human S9 fractions than in the presence of the rat S9 fraction. On the other hand, the other compounds (25%), including some aromatic amines and nitrosamines, showed a more potent mutagenicity in the presence of either one of the two human S9 fractions than in the presence of the rat S9 fraction. These data strongly suggest that the use of human liver S9 fraction in mutagenicity testing systems may be useful for a better understanding of the mutagenic effects of chemicals on humans.