Relationship between FTC 'tar' and urine mutagenicity in smokers of tobacco-burning or Eclipse cigarettes

The US Federal Trade Commission (FTC) classifies domestic cigarettes into one of three 'tar' categories based on 'tar' and nicotine levels. The objective of the present study was to determine urine mutagenicity in groups of smokers of ultra-low 'tar' (ULT), full-flavor low 'tar' (FFLT) and full-flavor 'tar' (FF) filtered cigarettes after switching to primarily tobacco-heating Eclipse cigarettes. Sixty-seven smokers maintained a specified diet and consumed ad libitum their usual brands of cigarettes, switched to Eclipse, and switched back to their usual brands. Twenty-four hour urine samples were collected weekly, concentrated on XAD-2 resin, and tested in the Ames mutagenicity assay using bacterial strains TA98 and YG1024 with S9 metabolic activation. Daily consumption of cigarettes was not significantly different (at P<0.05) between FTC 'tar' categories and average daily cigarette consumption did not change significantly in any smoker group after switching to Eclipse cigarettes. Average urine mutagenicity was 47% less (P<0.05) for ULT than for FFLT usual brand smokers as measured by the more sensitive strain YG1024, although no significant differences (P<0.05) were observed in urine mutagenicity between usual brand FTC 'tar' categories as measured by strain TA98. The reduction in urinary mutagens in the more sensitive strain, YG1024, observed in ULT smokers as compared with higher 'tar' categories suggest reduced exposure to mutagens. Usual brand salivary cotinine in the ULT group was significantly lower (P<0.05) than the FF group and the FFLT group. Salivary cotinine did not differ significantly (at P<0.05) among the smoker groups when smoking Eclipse compared to usual brand. After switching to Eclipse, the following reductions in urinary mutagenicity were observed: ULT, 70.1+/-6.4% (TA98), 70.9+/-6.2% (YG1024); FFLT, 77.1+/-2.4% (TA98), 73.6+/-2.0% (YG1024); and FF, 76.1+/-3.5% (TA98), 71.4+/-4.0% (YG1024). Across all 'tar' categories, cigarette smokers experienced significant reductions (P<0.05) in urine mutagenicity, but not salivary cotinine, upon switching to Eclipse. The reduction in urine mutagenicity when smoking Eclipse provides supporting evidence that Eclipse may present less risk of cancer compared to cigarettes currently in the market.     

Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes

Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamineO-acetyltransferase (OAT) activity. We have demonstrated that this strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes. In this paper, we compared the sensitivities of YG1024 with those of S. typhimurium YG1012, which has about 4 times higher OAt activity than YG1024 but lacks plasmid pKM101. It turned out that YG1024 was more sensitive to the mutagenic actions of 1-aminonaphthalene, 1-nitropyrene, 1,8-dinitropyrene and 2-nitronaphthalene than YG1012 and showed comparable sensitivity to 2-hydroxyacetylaminofluorene, 2-aminoanthracene and 2-amino-6-methyldipyridol[1,2-α:3′,2′-d]imidazole (Glu-P-1) to YG1012. These results suggested that YG1024 is more suitable than YG1012 for the efficient detection of mutagenic aromatic amines and nitroarenes.     

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

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

Phenotypic instability of Salmonella strain YG1024 during mutagenicity assays of arylamine promutagens

During spot tests using Salmonella TA98 derivatives (YG1021, YG1021) and TA100 derivatives (YG1026, YG1029), a unique response of O-acetyltransferase (OAT)-enhanced strains YG1024 and YG1029 to arylamines was observed. On plates containing rat-liver S9, these strains yielded revertant colonies induced in two separate concentric rings around the site of application, while the parent (TA98, TA100) and nitroreductase-enhanced strains (YG1021, YG1026) did not exhibit this response. The inner ring of revertants was accompanied by cytotoxicity and microcolony formation, with the outer ring in a region without background lawn toxicity. Addition of tetracycline to the top agar eliminated formation of the inner ring of YG1024 revertants in spot tests and reduced the revertant count in preincubation assays at cytotoxic dose levels of 2-aminoanthracene, 2-aminofluorene, 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline. Tetracycline sensitivity indicates that mutant colonies developing at high concentration/toxicity arose, in effect, from TA98 regenerated by functional loss of the tetracycline-resistance plasmid (pYG219) from YG1024. Mutant colonies found at low concentration/toxicity arose from normal plasmid-bearing YG1024. These results indicate the need to consider coincidental toxicity-induced instability in YG1024 during quantitative mutagenicity assays of arylamines and uncharacterized complex mixtures.     

Role of bacterial nitroreductase and O-acetyltransferase in urine mutagenicity assay of rats exposed to 2,4,6-trinitrotoluene (TNT)

The urine mutagenicity of rats exposed to 2,4,6-trinitrotoluene (TNT) by i.p. injection was studied in the Salmonella assay using indicator strains with various levels of 'classical' nitroreductase or acetyl-CoA:N-hydroxylarylamine O-acetyltransferase activity. The strains used were the conventional Salmonella typhimurium TA98, nitroreductase-deficient TA98NR and -overproducing YG1021, and O-acetyltransferase-deficient TA98/1,8-DNP6 and -overproducing YG1024. TA98, YG1021 and YG1024 clearly detected the increase of direct urine mutagenicity. A slight increase of mutagenicity was also detected with metabolic activation in YG1021 and YG1024. High levels of both nitroreductase and O-acetyltransferase significantly increased the sensitivity of the indicator strain to the mutagenicity of urine caused by TNT exposure, while the nitroreductase- or O-acetyltransferase-deficient strains gave negative responses.     

Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities

Salmonella typhimurium YG1021, YG1024, YG1026 and YG1029 are new derivatives of the Ames tester strains TA98 and TA100, with elevated 'classical' nitroreductase or acetyl-CoA:N-hydroxyarylamine O-acetyltransferase level. Thirty mutagens with different structures were tested using these strains and the sensitivities were compared with those of the conventional strains and of the enzyme-deficient strains. Elevated O-acetyltransferase activity of the indicator strains specifically increased their ability to detect the mutagenicity of aromatic nitro, amino and hydroxylamino compounds, whereas the strains with high nitroreductase activity were very sensitive to some nitroaromatics. The combined use of the isogenic tester strains with different metabolic capacities was quite useful to assess the intracellular metabolic activation and detoxification mechanisms of chemical mutagens.     

Evaluation of nitroreductase and acetyltransferase participation in N-nitrosodiethylamine genotoxicity

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

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

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

The influence of automobile exhausts on mutagenicity of soils: contamination with, fractionation, separation, and preliminary identification of mutagens in the Salmonella/reversion assay and effects of solvent fractions on the sister-chromatid exchanges in human lymphocyte cultures and in the in vivo mouse bone marrow micronucleus assay

To test the assumption that automobile exhausts contribute to soil mutagenicity, two soils with low levels of mutagenic activities were exposed to traffic exhausts at a heavily charged junction of German motorways (Autobahnen) for 3, 7, 10, 13, 17, 21, and 26 weeks. Indeed, in the presence of a metabolic activation system from rat liver (S9), an average increase of 8 and 9 (4 and 12) revertants per gram per week was found in Salmonella typhimurium TA 98 (TA 100). In the absence of S9, meaningful measurements were impossible on account of a concurrent dose dependent increase of toxicity. No correlation between the increase of mutagenicity and the contents of polycyclic aromatic hydrocarbons (PAH) could be detected. In another series, soils sampled at the roadside and at distances of 10 and 50 m of five roads near Mainz expressed 10–20-fold higher mutagenicity (revertants per gram) under identical test conditions as compared with the average of agricultural soils. Toxic effects, however, again confounded the results and no correlation between the distance from roads and the levels of mutagenicity could be demonstrated. Subsequently, Soxhlet-extraction with the solvent sequence dichloromethane, acetone, and toluene/diethylketone was found to be an optimum procedure for soils at roadsides. The mass balance of solvent fractionation of such soils revealed that <2% each belonged to organic acids and bases, 4% to fractions designed polar neutrals, 8% to polar aromatics, 7% to dichloromethane solubles, and 79% to cylohexane solubles, among them 63% acetone soluble compounds. The major part of mutagenicity (55–65%) was present in the fraction of polar aromatics, followed by polar neutrals and the acetone subfraction of cyclohexane solubles (10% each) summarizing the results obtained with S. typhimurium TA 98, TA 98NR, YG 1021, YG 1024, TA 100, YG 1026, and YG 1029 with and without addition of S9. The modified tester strains, either deficient in nitroreductase (TA 98NR) or overproducing nitroreductase (YG 1021, 1026) or O-acetyl-transferase (YG 1024, 1026), indicated a major contribution of nitroarenes to soil mutagenicity. With respect to mutagenic PAH, high pressure liquid chromatography (HPLC) revealed that >90% of dibenz[a,h]anthracene (4.18 mg/kg soil), benzo[a]pyrene (1.96 mg), benzofluoranthenes (0.14 mg), and benz[a]anthracene (0.18 mg) were present in the acetone subfraction of cyclohexane solubles. Concentrations and mutagenic activities, however, did not correlate. Additional preparative and analytical HPLC of the solvent fractions of polar neutrals and polar aromatics, resulted in the tentative identification of 2-nitrofluorene. Analysis of the vertical profile of soil revealed an increase of mutagenicity per gram from the surface to a maximum at 5–15 cm depth and a subsequent decrease with very little activity remaining deeper than 35 cm. In human lymphocyte cultures, the fraction of polar aromatics, 0.01–0.3 μg/ml, induced 11.27±4.76–20.70±6.19 sister-chromatid exchanges (SCE) per cell in the absence of S9 (solvent control: 10.16±4.83 SCE per cell) and 12.77±6.53–17.87±4.93 SCE per cell in the presence of S9 (solvent control: 8.37±3.92 SCE per cell). However, no activities could be detected in the fractions of polar neutrals and non-polar neutrals. Again, negative results were obtained in the in vivo mouse bone marrow micronucleus assay at 2000 mg/kg p.o. with all fractions.     

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

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

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

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

Specificity and sensitivity of Salmonella typhimurium YG1041 and YG1042 strains possesing elevated levels of both nitroreductase and acetyltransferase activity

Acetyltransferase and nitroreductase are enzymes involved in the intracellular metabolic activation of nitroarenes and/or aromatic amines in Salmonella typhimurium. The plasmid carrying both the acetyltransferase and nitroreductase genes was introduced into S. typhimurium TA98 and TA100. The resulting strains, YG1041 and YG1042, respectively, showed high levels of both enzyme activities and were more sensitive to the mutagenic action of some nitro-aromatic compounds such as 2-nitrofluorene, 1-nitropyrene and p-nitrophenetole than did the sensitive strains previously established in this laboratory or the conventional strains. These results indicate that the new strains permit the very efficient detection of the mutagenicity of nitroarenes in the environment.     

Differential responses of N-nitrosoamines and aromatic amines in the plant cell/microbe coincubation assay

The plant cell/microbe coincubation assay is based on employing living tobacco cells in suspension culture as the activating system for promutagens and the Ames/Salmonella cells as the genetic indicator system. In contrast to aromatic amines(e.g. 2-aminofluorene andm-phenylenediamine) that were previously reported to be activated to products mutagenic in theS. typhimurium strains TA98 or YG1024 by tobacco cells, promutagenic N-nitrosoamines (N-nitrosodimethylamine, N-nitroso-morpholine, N-nitrosopiperidine, N-nitrosomethyl-2-hydroxypropylamine) were not activated to product(s) mutagenic inS. typhimurium TA 100.     

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

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

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

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.     

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

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

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

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

Mutagenic activity of airborne particulate matter in a petrochemical industrial area

Exposure to airborne particulate matter has adverse effects on human health and ecosystem. Mutagenic activity of airborne particulate organic matter extracts in three time periods from total suspended particles (TSP) and particles less than 10 μm (PM10) was evaluated in an area under the influence of a petrochemical industry located in the town of Triunfo, Brazil. The extracts were investigated using the Salmonella/microsome assay, with the microsuspension method. The extracts were obtained by sonication extracted using dichloromethane (DCM) solvent. The fractions were tested for mutagenicity with the Salmonella typhimurium strains TA98 (with and without metabolic activation), TA98NR and TA98/1,8DNP₆; or YG1021 and YG1024. A positive frameshift mutagenic response was observed for the environmental samples during the different periods. The responses according to percentage of extractable organic matter (EOM%), EOM/m³, revertants/μg (rev/μg) and revertants/m³ (rev/m³) were lower for TSP than for PM10 extracts. The highest rev/m³ values were observed in PM10 extract samples collected in winter, July 2005, in the presence (13.79 rev/m³) or absence (6.87 rev/m³) of S9 fraction. Similarly in the first (1995) or second period (2000) the highest values for TSP were observed in winter, but with lower activity (3.00 and 0.89 rev/m³ respectively). The responses observed for the nitrosensitive strains suggest the contribution of nitro, amino and/or hydroxylamino derivatives of PAHs to the total mutagenicity of matter extracted from airborne particles. The Salmonella/microsome assay was a sensitive method to define areas contaminated by genotoxic compounds, even in samples with TSP or PM10 values that are acceptable according to legal environmental quality standards, favoring environmental control measures with an effective response seen in the population's improved quality of life.     

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<2,2′-dimethyl-5,5′-dinitroazoxybenzene (2,2′-DM-5,5′-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB), and aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<4HA2NT4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2,6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2′-dimethyl-3,3′-dinitroazoxybenzene (2,2′-DM-3,3′-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.     

Sensitive method for the detection of mutagenic nitroarenes and aromatic amines: new derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyltransferase levels

Acetyl-CoA: N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the intracellular metabolic activation of arylhydroxylamines derived from mutagenic nitroarenes and aromatic amines. The acetyltransferase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1024 and YG1029) had about 100 times higher 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole (N-hydroxy-Glu-P-1) O-acetyltransferase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic actions of 2-nitrofluorene, 1-nitropyrene, 1,8-dinitropyrene, 2-amino-6-methyldipyrido[1,2-a:3',2-d)-imidazole (Glu-P-1), 2-aminofluorene and 2-aminoanthracene. These results indicate that the new strains permit the efficient detection of the mutagenicity of environmental nitroarenes and aromatic amines.