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.     

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.     

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.     

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.     

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.     

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.     

Structural activity relationship between Salmonella-mutagenicity and nitro-orientation of nitroazaphenanthrenes

Nitroazaphenanthrenes (NAphs) and their N-oxides (NAphOs) were synthesized as derivatives with nitrogen atoms in the 1, 4, and 9 positions of phenanthrene rings, and as nitrated derivatives substituted at the 1, 2, 3, 4, 5, 6, 7, and 8 positions of phenanthrene rings. To determine the structure activity relationship of these derivatives, all 19 isomers were bioassayed with Salmonella tester strains. NAphs substituted at the 4, 6, 7 and 8 positions were mutagenic for TA98, and 1-, 2-, and 3-N-9-AphOs, 6-N-1-AphO and 6-N-4-AphO were mutagenic for TA98 and TA100 without the S9 mix, while 5-N-1-AphO and 5-N-9-AphO were non- or weakly mutagenic. Nitrated derivatives, 6-N-4-Aph, 6-N-9-Aph, 6-N-1-AphO, and 6-N-4-AphO, were powerful mutagens for TA98 and TA100. Mutagenicity was enhanced by mutant strains producing nitroreductase, such as YG1021 and 1026, and by those producing O-acetyltransferase, such as YG1024 and 1029. Nitro derivatives substituted at positions 4 and 5 in the phenanthrene rings were perpendicular, while those at positions 2, 3, 6 and 7 were coplanar to the phenanthrene rings. NAphs substituted at the 1 and 8 positions were noncoplanar due to steric hindrance of the aromatic proton at the peri position. On the other hand, 1,5- and 1,8-dinitro-4-azaphenanthrenes showed high mutagenicity for strains TA98 and TA100 in the absence of the S9 mix, and were strongly enhanced by nitroreductase and O-acetyltransferase, over-producing mutants. Therefore, it was found that the mutagenic potency of NAphs and NAphOs was closely associated with the chemical properties and orientation of nitro substitution of aromatic rings.     

Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

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.     

Mutagenic characteristics of river waters flowing through large metropolitan areas in North America

The hanging technique using blue rayon, which specifically adsorbs mutagens with multicyclic planar structures, has the advantages over most conventional methods of not having to bring large volumes of water back to the laboratory for extraction of organic materials. Therefore, for the same effort the hanging blue rayon technique allows for the analysis of more samples from remote sites, although it has a disadvantage of not allowing quantitative analysis. In this study, the blue rayon hanging technique was used to collect organic mutagens in river waters that flow through metropolitan areas in northeastern North America. Monitoring was performed at a total of 21 sites: the Providence River system (4 sites), the Charles River (2 sites), the Potomac River (6 sites), the St. Lawrence River (5 sites), the Hudson River (3 sites), and the East River (1 site). Mutagenicity was evaluated using the Salmonella assay with strains TA98, TA100, YG1024, YG1041, and YG1042 with and without metabolic activation. The results demonstrated that strains YG1041 and YG1024 were much more sensitive than TA98 with S9 mix. Fifteen samples out of 21 were positive in YG1041 with S9 mix. Six samples gave 5000-18,400 revertants/g blue rayon equivalent. YG1042 was also much more sensitive than TA100. Eight samples were positive in YG1042 with S9 mix. The highest activity was 10,200 revertants/g blue rayon equivalent. The overall results showed that rivers flowing through major cities in North America contain frameshift-type, aromatic amine-like mutagenic activity. However, the levels of mutagenic activity in these rivers were much lower than expected based on prior analyses and calculated population-to-discharge ratios. Further research, such as detailed chemical analyses and/or simultaneous comparisons of several different adsorbents (e.g. XAD and blue rayon), will be needed to clarify the observed differences between North American blue rayon values and published values for European and Asian river systems.     

Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy-resin hardeners

3 epoxy-resin hardeners, 4,4'-diaminodiphenyl ether (DDE), 4,4'-diaminodiphenylmethane (DDM), and 4,4'-diaminodiphenylsulfone (DDS), and their N-acetyl and N,N'-diacetyl derivatives were examined for their mutagenicity using Salmonella typhimurium TA98 and TA100 as the tester stains and an S9 mix containing a rat-liver 9000 X g supernatant fraction as the metabolic activation system. DDE and DDM were mutagenic towards TA98 and TA100 in the presence of S9 mix while DDS exhibited no significant mutagenic activity towards these tester strains. These epoxy-resin hardeners were metabolized in vivo and their N-acetyl and N,N'-diacetyl metabolites were found in the urine. Among these acetyl metabolites, only N-acetyl-DDE was found to be mutagenic towards TA98 and TA100 in the presence of S9 mix. None of these acetyl metabolites exhibited significant mutagenic activity towards these tester strains in the absence of S9 mix.     

Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes

We have reviewed the mutagenicity of benzidine analogues (including benzidine-based dyes), with a primary emphasis on evaluating results of the Salmonella/microsome mutagenicity assay. Many of these amines are mutagenic in tester strains TA98 and TA100 but require exogenous mammalian activation (S9) for activity. A few amines with halogen or nitro-groups in the structure are direct-acting mutagens. The addition of a sulfonic acid moiety to the molecule of benzidine reduced the mutagenicity of benzidine; whereas, methoxy, chloro, or methyl group additions did not. Complexation with a metal ion also decreased the mutagenicity. A substitution of an alkyl group on the ortho position next to an amine group also influenced the mutagenicity. Most carcinogenic benzidine analogues are mutagenic, and their metabolism to electrophiles that interact with DNA, leading to mutations, plays a central role in their carcinogenesis.     

Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates

The mutagenic potential of three alkyl 2-cyanoacrylate adhesives, three commercial alkyl 2-cyanoacrylate adhesives and three methyl 2-cyano-3-phenylacrylates, was assessed using the Salmonella/microsome mutagenicity assay. Compounds were tested with and without Aroclor 1254-induced rat-liver homogenate (S9 mix). The methyl 2-cyanoacrylate adhesives were mutagenic in the standard plate test with S. typhimurium strain TA100 with and without S9 activation. Methyl 2-cyano-3-(2-bromophenyl)acrylate revealed a direct mutagenic action to S. typhimurium strain TA1535. The compounds most toxic towards the bacterium S. typhimurium, were the methyl 2-cyanoacrylate adhesives (greater than 500 micrograms/plate). All alkyl 2-cyanoacrylate adhesives were tested in a modified spot test for volatile compounds with tester strain TA100. Mutagenic and toxic effects were observed with the three methyl 2-cyanoacrylate adhesives. It can be concluded from the results that the bacterial toxicity and mutagenicity of methyl 2-cyanoacrylate adhesives may be due to the methyl 2-cyanoacrylate monomer.     

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.     

Relationship between mutagenic potency in Salmonella typhimurium strains and the chemical structure of nitro biphenyls

Most of the positional isomers of mono-, di-, tri- and tetranitrobiphenyls were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8DNP6 in the absence of S9 mix. In mono- and dinitrobiphenyls, the structure requirements favoring mutagenic activity are the presence of a nitro group at the 4-position and its absence at the 2-position. TA98 and TA98/1,8DNP6 were reverted by 2-position-free 4-nitro analogues, but TA98NR was not reverted. The results suggest that direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes. Some of the tri- and tetranitrobiphenyls e.g. 3,4,3'-, 3,4,4'-, 3,4,3',4'- and 3,4,2',4'-derivatives reverted not only TA98 and TA98/1,8DNP6 but also TA98NR. Those derivatives commonly have 2 nitro groups at an adjoining position (3,4-dinitro group), whereas 2,4,2',4'-tetranitrobiphenyl, which has strong potency not only in TA98 and TA98/1,8DNP6 but also in TA98NR, possesses 2 nitro groups at the 2-position of each benzene ring.     

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.     

Effect of various alkyl and unsaturated substituents on the mutagenicity of some nitrophenyl thioethers.

A variety of nitro-substituted phenyl alkyl/aryl thioethers and nitroso-substituted phenyl alkyl/aryl thioethers have been synthesized and tested for their mutagenicity towards Salmonella typhimurium strain TA100, TA98, TA98NR and TA98/1,8-DNP(6) in the absence of S9 mix. The relative order of mutagenicity in TA98 and TA100 among p-nitrophenyl thioethers having alkyl or aryl substituents is allyl>phenyl>benzyl>butyl>propyl>ethyl>methyl. Compounds having an alkyl chain C(6) to C(12) were found to be non-mutagenic. Among the various positional isomers (ortho, meta and para) of nitro-substituted diphenyl thioethers only the compounds having the -NO(2) function at the para position is mutagenic, whereas compounds having a -NO(2) function at ortho and meta are non-mutagenic. However, the reduced intermediate, ortho-nitroso derivative was found to be mutagenic in all the four strains but the meta-nitroso derivative was found to be non-mutagenic. All mutagens were found to be non-mutagenic when tested in nitroreductase deficient strain TA98NR, whereas their nitroso intermediates are found to be mutagenic. A substantial fall in the mutagenic activity is observed when some mutagens are tested in O-acetyltransferase deficient strain TA98/1,8-DNP(6).     

Mutagenicity of nitro- and amino-substituted phenazines in Salmonella typhimurium

The nitro- and amino-substituted phenazines were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98 and TA98NR. Of 7 tested nitrophenazines, 4 were mutagenic in the absence of a microsomal metabolic activation system (S9 mix) and were more mutagenic in TA98 than in TA98NR. The order of mutagenicity of nitrophenazines in TA98 is 1.7- less than 2- less than 2.8- less than 2.7-substituted phenazine. Of 7 tested amino derivatives, 4 exhibited mutagenic activity with S9 mix in TA98. 1-Nitro-, 1-amino, 1.6-dinitro-, 1.9-dinitro-, 1.6-diamino- and 1.9-diamino-phenazine were not mutagenic. As regards the relationship between mutagenic potency and chemical structure of the phenazines, the results suggested that structural requirements favoring mutagenic activity were the presence of substituents at the 2 and/or 7 position. Furthermore, 2.7-disubstituted phenazines were extremely mutagenic, 2.7-dinitrophenazine and 2.7-diaminophenazine induced 36,450 and 12,110 rev./nmole, respectively. In the preliminary study, 2.7-diaminophenazine was identified by gas chromatography/mass spectrometry from the reaction mixture of m-phenylenediamine and hydrogen peroxide.     

Mutagenicity of 2,6-dinitrotoluene and its metabolites, and their related compounds in Salmonella typhimurium

The mutagenic activities of 2,6-dinitrotoluene (2,6-DNT) and its 6 metabolites, and their 8 related compounds were examined using Salmonella typhimurium strains TA98 and TA100 in the absence or presence of S9 mix. 2,6-DNT itself showed no mutagenicity toward either strain, but 2,6-dinitrobenzaldehyde (2,6-DNBAl), one of the metabolites of 2,6-DNT, showed the highest mutagenic activity in strain TA100. 2,6-DNBAl was a direct-acting mutagen, not requiring metabolic activation. The other compounds containing nitro groups showed weak or no mutagenic activity. This result suggests that the direct-acting mutagenicity of 2,6-DNBAl is mainly due to the aldehyde group of the 2,6-DNBAl molecule.     

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.