Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

Mutagenicity of processed bidi tobacco: possible relevance to bidi industry workers

The genotoxic potential of bidi tobacco was evaluated by mutagenicity testing of aqueous, aqueous: ethanolic, ethanolic and chloroform extracts of processed tobacco used in the manufacture of 'bidis', indigenous forms of cigarettes smoked in India. The Salmonella/mammalian microsome test (Ames assay) was used to detect mutagenicity in tester strains TA98, TA100 and TA102. The extracts were tested in the absence and presence of metabolic activation using liver S9 from rat and hamster, and following in vitro nitrosation with sodium nitrite at acidic pH. All the extracts were non-mutagenic in the absence of nitrosation. The nitrosated aqueous extract was mutagenic in strains TA98 and TA100. While weak mutagenicity was elicited by the nitrosated aqueous: ethanolic extract in TA100, the nitrosated ethanolic extract induced a 3-fold increase in the number of revertants in the same strain. Moreover both these extracts elicited a strong mutagenic response in TA102, while the chloroform extract was non-mutagenic even after nitrite treatment. The present study indicates that workers employed in the bidi industry are exposed to potentially mutagenic and genotoxic chemicals in the course of their occupation.     

Formation of mutagens by amino-carbonyl reactions

The formation of mutagens by amino-carbonyl reactions of 20 kinds of amino acid and sugars after heating at 100 degrees C for 10 h was examined by the Ames test. The browned solutions of Gly, Ala, Val, Leu, Ile, Ser, Thr, Gln, Lys X HCl, Arg, Phe, Cys, Met and Pro with Glc caused mutation of Salmonella typhimurium TA100 and/or TA98 with or without S9 mix. The presence of S9 mix increased the mutagenic activity of the browned solutions of Cys and Phe with Glc on TA100 and of those of Gly, Ala, Val, Ile and Cys on TA98, but decreased the activity of other solutions. No revertants of Salmonella were induced by the browned solutions of Trp, Tyr, Asp, Asn, Glu and (Cys)2 with Glc. Among positive browned solutions, Cys, Lys, Arg and Phe had the stronger activity, but their activity was weak compared with that of pyrolysates or chemical mutagens such as Trp-P-1, Trp-P-2 and 4-nitroquinoline-N-oxide. The mutagenic activity of the browned solutions increased with prolongation of heating time and varied with the pH of the reaction mixture. Fru, Gal, Ara, Xyl, Man, Lac and Suc also had the ability to form mutagens in the browning reactions with amino acids.     

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.     

Salmonella/mammalian microsome assay with tetranitromethane and 3-nitro-L-tyrosine

The nitrosating agent tetranitromethane (TNM) and the nitrosation product 3-nitro-L-tyrosine (NT) were tested for mutagenic activity in the Salmonella/mammalian microsome assay. TNM showed strong genotoxic activity: it was mutagenic in all tester strains used (TA97, TA98, TA100, and TA102). The maximum mutagenic activity was reached between 16 and 32 micrograms/plate using the standard plate test; higher amounts led to distinct bactericidal effects. The mutagenicity was independent of an in vitro activation system. In the preincubation assay an increased bactericidal effect was observed. In contrast to TNM, NT, the nitrosation product, was non-mutagenic and non-toxic in the standard plate test and with the preincubation method up to 5000 micrograms/plate with and without S9 mix and with all tester strains used. Although TNM is a strong direct-acting mutagen, its nitrosating effect on proteins does lead to nongenotoxic nitro products of tyrosine in proteins.     

Mutagenic action of a series of epoxides.

The mutagenicity of a series of 13 epoxide compounds was studied using a bacterial plate assay system. The histidine-dependent tester strains TA98 (for frameshift mutagens) and TA100 (for base-pair substitution mutagens) of Salmonella typhimurium were used. Mutagenicity was evaluated both with and without the additon of rat liver microsomal extract. Dieldrin, diglycidyl ether of bis phenol A and 3 of its homologues were not mutagenic. Allyl glycidyl ether, n-butyl glycidyl ether, vinly cyclohexene diepoxide, glycidol, glycidal-dehyde, diglycidyl ether, diepoxybutane and diglycidyl ether of substituted glycerine were mutagenic in the TA100 strain, causing reversion of the bacteria to histidine independence. Dose-reponse curves of the mutagenicity of the latter 4 compounds were obtained. On a molar basis, glycidaldehyde was about 20-50 times more potent in producing mutation that were the other 3 epoxides in the dose-response test. In general, the mutagenicity of the epoxides was not enhanced or diminished by the addition of microsomal extract.     

Soil mutagens are airborne mutagens: variation of mutagenic activities induced in Salmonella typhimurium TA 98 and TA 100 by organic extracts of agricultural and forest soils in dependence on location and season

As our hypothesis was that soil mutagens are airborne mutagens, possibly modified by soil microorganisms, we checked solvent extracts from agricultural and forest soils collected during late summer in the environment of Mainz, a region highly charged by anthropogenic air pollution, or near Bayreuth, a rural low charged region of Germany, or in a remote region of western Corsica without anthropogenic air pollution for the presence of mutagenicity in Salmonella typhimurium. Levels of mutagenic activities were quantified by calculation of revertants/g from the initial slope of dose-response curves applying tester strains S. typhimurium TA 98 and TA 100 in the absence and presence of an activation system from rat liver (S9). Three soils from Corsica did not induce mutagenicity under any test condition. However, most soils from Germany exhibited mutagenic activities, though preferentially in strain TA 98, but no statistically significant differences could be detected between 27 soils from the Mainz and nine soils from the Bayreuth regions. On the other hand, no correlation could be detected between the levels of mutagenic activities at any test condition and agricultural practice - rye growing, viniculture, fruit growing, meadow, and fallow - texture of soils - % composition of clay, slit, and sand - or the contents of organic matter. The only significant difference of mutagenicity was, however, found with S. typhimurium TA 98-S9 between forest soils of pH approximately 4.0 as compared with agricultural soils of pH approximately 7.0. The presence of antimutagens in soil as demonstrated by the course of dose-response curves of the three soils from Corsica may be another possible confounder. Calculation of mean values of mutagenic activities for all soils from Germany gave the following results: S. typhimurium TA 98: 69.7+/-153.2 (-S9); 63.0+/-176.3 (+S9); S. typhimurium TA 100:-144.7+/-399.4 (-S9); 43.3+/-172.0 (+S9) revertants/g of dry soil. In another series of experiments, soil mutagenicity in 10 rye fields near Mainz was monitored for 1 year. It became evident that low levels of mutagenic activities in late summer increased during autumn, reached a peak in late winter, and subsequently, decreased during spring and summer. These results agree with the hypothesis of an airborne origin of soil mutagens, deposition, and an adjacent transformation to non-mutagenic compounds by soil microorganisms.     

Preferential induction of AT-TA transversion, but not deletions, by chlorambucil at the hisG428 site of Salmonella typhimurium TA102.

The chemotherapeutic agent chlorambucil effectively induces deletion mutations in mouse germ cells. The possibility that this chemical also effectively induces deletion mutations in bacterial DNA was examined using Ames Salmonella tester strains. Chlorambucil was mutagenic only to strains TA102 (hisG428, rfa/pKM101) and YG2975 (hisG46, rfa/pKM101) when S9 mix was absent. Since strain TA102 can detect short deletions, the mutational changes of TA102 induced by this agent without S9 mix were directly determined by the DNA sequencing technique. It turned out that chlorambucil did not induce deletion mutations but preferentially induced AT-TA transversions at the hisG428 site of plasmid pAQ1 of strain TA102. These results caution that the positive results induced by chlorambucil in mutagenicity tests do not necessarily mean the occurrence of deletions.     

A new Salmonella tester strain,TA97, for the detection of frameshift mutagens: A run of cytosines as a mutational hot-spot

We have developed a new Salmonella tester strain, TA97, for use in the Salmonella/microsome mutagenicity test. DNA sequencing has shown that this strain contains and added cytosine, resulting in a run of six cytosines at the mutated site in the histidine D gene. Its mutagenic specificity is similar to that of the frameshift mutagen tester strain, TA1537, which also contains an added cytosine in a run of cytosines and is currently among the five standard tester strains used for general mutagen screening. We assessed the mutagenic potency of 21 frameshift mutagens for TA1537 and TA97. TA97 was considerably more sensitive than TA1537 to reversion by these frameshift mutagens. In addition, one agent, PR toxin (from Penicillium roqueforti), which was not detected by any of the previously existing standard tester strains, did revert TA97; and two substituted aryl-alkyl triazenes, which had not been reported previously to be frameshift mutagens, were mutagenic in this new tester strain. We suggest that TA1537 be replaced by TA97 for general screening of mutagenicity.     

Evaluation of the mutagenic potential of endosulfan using the Salmonella/mammalian microsome assay

The mutagenic potential of endosulfan, a polychlorinated insecticide, was assessed using the highly sensitive Salmonella tester strains TA97(a), TA98, TA100 and TA102. It exhibited a toxic effect at dose levels of 50 micrograms/plate and higher. Plate incorporation studies did not show mutagenic response with any of the tester strains used. A modification of the assay using a preincubation procedure showed mutagenic activity with and without metabolic activation with TA97(a) only. Increased toxicity was observed after addition of phenobarbital-induced S9 mix.     

Response of the L-arabinose forward mutation assay of Salmonella typhimurium to frameshift-type mutagens

The present study was designed to evaluate the capacity of the L-arabinose resistance test of Salmonella typhimurium in the detection of frameshift-type mutagens. To this end the response of the Ara test was examined with respect to 15 chemicals which had been previously described as able to revert the Ames tester strain TA97. The mutagenicity of each compound was determined by the liquid test under experimental conditions which optimize the mutagenic response of the Ara test with the tester strain BA9. Strain TA97 was used simultaneously with BA9. The Ara forward-mutation assay efficiently detected the mutagenic activity of 14 out of the 15 chemicals assayed. PR toxin was the only compound which gave a weak dose response without doubling the spontaneous mutant level. In comparison with the Ara test, a total of 3 chemicals (HZ, PE and PR toxin) were not found to be mutagenic with strain TA97. In most cases (11/15) the mutagenic response of the Ara test was comparatively greater than that of strain TA97. Three chemicals (DEO, PRF and 9-AA) were detected with quite similar degrees of sensitivity by both mutation assays. ICR-191, which seems highly specific in reverting frameshift mutations with added cytosines in a run of cytosines, was the only chemical with a lower mutagenic activity in the Ara test than in strain TA97. The results enhance the interest of the L-arabinose forward-mutation assay as an alternative to the set of specific tester strains used by the histidine reverse-mutation assay in massive, general and primary screening for genotoxic agents.     

Mutagenicity studies of methyl-tert-butylether using the Ames tester strain TA102

Methyl-tert-butylether (MTBE) is an oxygenate widely used in the United States as a motor vehicle fuel additive to reduce emissions and as an octane booster [National Research Council, Toxicological and Performance Aspects of Oxygenated Motor Vehicle Fules, National Academy Press, Washington, DC, 1996]. But it is the potential for MTBE to enter drinking water supplies that has become an area of public concern. MTBE has been shown to induce liver and kidney tumors in rodents but the biochemical process leading to carcinogenesis is unknown. MTBE was previously shown to be non-mutagenic in the standard Ames plate incorporation test with tester strains that detect frame shift (TA98) and point mutations (TA100) and in a suspension assay using TA104, a strain that detects oxidative damage, suggesting a non-genotoxic mechanism accounts for its carcinogenic potential. These strains are deficient in excision repair due to deletion of the uvrB gene. We hypothesized that the carcinogenic activity of MTBE may be dependent upon a functional excision repair system that attempts to remove alkyl adducts and/or oxidative base damage caused by direct interaction of MTBE with DNA or by its metabolites, formaldehyde and tert-butyl alcohol (TBA), established carcinogens that are mutagenic in some Ames strains. To test our hypothesis, the genotoxicity of MTBE-induced DNA alterations was assayed using the standard Ames test with TA102, a strain similar to TA104 in the damage it detects but uvrB + and, therefore, excision repair proficient. The assay was performed (1) with and without Aroclor-induced rat S-9, (2) with and without the addition of formaldehyde dehydrogenase (FDH), and (3) with human S-9 homogenate. MTBE was weakly mutagenic when tested directly and moderately mutagenic with S-9 activation producing between 80 and 200 TA102 revertants/mg of compound. Mutagenicity was inhibited 25%-30% by FDH. TA102 revertants were also induced by TBA and by MTBE when human S-9 was substituted for rat S-9. We conclude that MTBE and its metabolites induce a mutagenic pathway involving oxidation of DNA bases and an intact repair system. These data are significant in view of the controversy surrounding public safety and the environmental release of MTBE and similar fuel additives.     

Collaborative study of mutagenicity with Salmonella typhimurium TA102.

Thirty compounds of various chemical classes were investigated for mutagenicity in a collaborative study (3 laboratories) using Salmonella typhimurium TA102. With 5 compounds, namely hydrazine sulfate, phenylhydrazine, hydralazine, glutardialdehyde and glyoxal, mutagenicity was detected by all laboratories. Formaldehyde was assessed as weakly mutagenic in only 1 of 3 laboratories. The remaining 24 agents were uniformly described as non-genotoxic in TA102. In spite of the overall good qualitative agreement in the mutagenicity results between the 3 laboratories some quantitative discrepancies occurred in the dose response of the mutagenic compounds. Varying inter- and intra-laboratory differences in the spontaneous rate of revertants were obtained. The usefulness of the tester strain TA102 in routine mutagenicity testing is discussed.     

Mutagens in coffee and tea.

Coffee prepared in the usual way for drinking contains a substance(s) that is mutagenic to Salmonella typhimurium TA100 without mammalian microsomal enzymes. One cup of coffee (200 ml) contains mutagen(s) inducing 1.4-4.6 X 10(5) revertants under standard conditions. Instant coffee too is mutagenic to TA100 and one cup of instant coffee prepared from 1 g of coffee powder and 200 ml of water induced 5.6-5.8 X 10(4) revertants of TA100. Caffeine-free instant coffee also has similar mutagenicity. Addition of microsomal enzymes abolished the mutagenicity. Black tea, green tea and Japanese roasted tea were also mutagenic to TA100 without S9 mix and one cup of these teas prepared in the ordinary way produced 1.7-3.8 X 10(4) revertants of TA100. Black tea and green tea were also mutagenic to TA98 in the presence of S9 mix after treatment with a glycosidase from Aspergillus niger, hesperidinase. This type of mutagen in one cup of black tea induced 2.4 X 10(5) revertants of TA98.     

Detection of mutagenic activity in automobile exhaust

Using the Ames Salmonella-microsome system, we detected mutagenic activity in the exhaust from two kinds of 4-cycle gasoline engines of unregulated and regulated cars, and from diesel engines, as well as in the particulates from air collected in tunnels. The mutagenicity of particulates from a car equipped with a catalyst (regulated car), as compared with that from an unregulated car, was reduced very much (down to 500 from 4500 revertants/plate/m3 in tester strain TA98). However, the mutagenicity of the ether-soluble acid and neutral fractions from the condensed water of emissions from a regulated car was still high (down to 2880 from 10 900 revertants/plate/m3 in tester strain TA100). The mutagenic activity of emission exhaust from old diesel car engines was very high; the particulates showed 9140 and 19 600 revertants/plate/m3 from strain TA98 incubated with an activating rat-liver S9 fraction. A small diesel engine of the type used for the generation of electric power or in farm machinery also produced exhaust with highly mutagenic particulates. The mutagenic activity of a methanol extract of particulate air pollutants collected in a highway tunnel showed 39 revertants/plate/m3 toward strain TA98 and 87 toward strain TA100. The ether-soluble neutral fraction yielded 86 revertants/plate/m3 from strain TA98 and 100 from strain TA100. This fraction also contained carcinogenic compounds, including benzo[a]pyrene, benzo[e]pyrene, benz[a]anthracene, benzo[ghi]perylene and chrysene. Very high mutagenic activity was detected, especially in the particulate air pollutants collected at night, in another tunnel on a superhighway: 60-88 revertants/plate/m3 from strain TA100 for the sample collected by day, but 121-238, by night. Night traffic includes many more diesel-powered vehicles compared with gasoline-powered automobiles.     

The effect of quercetin on the mutagenicity of 2-acetylaminofluorene and benzo[alpha]pyrene in Salmonella typhimurium strains

The comutagenic and desmutagenic effect of quercetin on the mutagenicity of typical mutagens e.g. 2-acetylaminofluorene (AAF), 4-nitroquinoline-1-oxide (4NQO) and benzo[alpha]pyrene (B[a]P), in Salmonella typhimurium TA98, TA100 and TA98/1,8 DNP6 were examined. In the mixed application of AAF with quercetin in the presence of mammalian metabolic activation system (S9 mix), the numbers of revertants in TA98 increased by as much 2.2-5.0-fold compared with the sum of those in the separate applications of AAF and quercetin. A 1.4-2.7-fold increase was observed in TA100. Quercetin did not affect the mutagenicity of 4NQO, and depressed that of B[a]P. Dose-response curves for mutagenicity of quercetin with or without AAF (5 micrograms/plate) were examined. The results suggest that quercetin, present in a molarity of up to 1.5 times that of AAF, is apparently effective in enhancing the mutagenicity of AAF, because a linear dose-response curve was observed in the range of 0-5 micrograms/plate quercetin with AAF although quercetin alone was not mutagenic in the same range. Dose-response curves for mutagenicity of quercetin with or without 5 micrograms/plate B[a]P did not increase compared with that for quercetin alone. The mutagenicity of the mixed application of B[a]P with quercetin was reduced to about 60% of the sum of separate application at doses ranging from 25 to 100 micrograms/plate of quercetin. Since enhancement and depression of mutagenicity by quercetin were observed for indirect mutagens, AAF and B[a]P, respectively, in the presence of S9 mix, quercetin may affect the metabolic pathway of these mutagens.     

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.     

Studies on the effect of ascorbic acid and selenium on the genotoxicity of nitrofurans: nitrofurazone and furazolidone

The genotoxic properties of nitrofurazone and furazolidone were studied using the Ames test and SOS-chromotest. Both compounds were found to act as strong mutagens on the TA97 and TA102 strains of S. typhimurium and to induce the SOS-repair system in the PQ37 strain of E. coli. A good concordance was found between the mutagenic activity and the ability to induce the SOS system. Ascorbic acid and sodium selenite only very slightly lowered the genotoxic effect of the 2 nitrofurans studied both in the Ames test and in the SOS-chromotest.     

Demonstration of a powerful mutagenic dinitropyrene in airborne particulate matter

Of the many nitroarenes, dinitropyrenes (DNPs) have the potential to revert Salmonella typhimurium his- mutants. This study was conducted to investigate the potential mutagens present in airborne particulate matter collected in Santiago, Chile. 5 organic substances extracted with dichloromethane showed mutagenic rates of from 38.9 to 287 revertants per m3 of air for S. typhimurium his- strain TA98 without S9 mix. 4 of the samples had greatly reduced mutagenicity for strain TA98/1,8DNP6 but not for strain TA98NR. The 1-nitropyrene (1-NP) content accounted for 0.06-0.15 microgram per g of particulate, as determined by high-performance liquid chromatography (HPLC), but the contribution of the compound to mutagenicity was less than 1% of the total activity. On the other hand, by using two columns in the HPLC, DNPs of 1,6- and 1,8-isomers were detected in the samples pooled after the determination of 1-NP, and the amount of the derivatives was about 0.2 microgram per g of particulate matter.