Metabolism of 2,4-dinitrotoluene by Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6, and mutagenicity of the metabolites of 2,4-dinitrotoluene and related compounds to strains TA98 and TA100

The products detected in the incubation of 2,4-dinitrotoluene (2,4-DNT) with Salmonella typhimurium strains TA98 and TA98/1,8-DNP6 were nitrosonitrotoluenes, hydroxylaminonitrotoluenes, aminonitrotoluenes and dimethyl dinitroazoxybenzene. The capacity of TA98NR to reduce 2,4-DNT was much lower than that of TA98 and TA98/1,8-DNP6. The bacterial products showed no mutagenic activity in the Ames assay using TA98 and TA100. These results indicate that the lack of mutagenic activity of 2,4-DNT is not due to low reductive metabolism of 2,4-DNT by the bacteria, but to the lack of mutagenic activity of the bacterial reductive products of 2,4-DNT, including dimethyl dinitroazoxybenzene.     

The mutagenic potencies of plant extracts containing quercetin in Salmonella typhimurium TA98 and TA100

Four commercial ethanolic plant extracts, Tinctura Alchemillae, Extractum Crataegi, Extractum Myrtilli and Tinctura Hyperici, were tested for their mutagenicity in Salmonella typhimurium TA98 and TA100 with and without S9 mix obtained from rats pretreated with phenobarbital. The extracts studied differed greatly in their mutagenic potencies but exhibited a very similar mutation pattern in which the strongest effect was always seen in tester strain TA98 with S9 mix. Simultaneously we investigated the extracts for the presence of quercetin and kaempferol. Only quercetin was detected in small amounts by thin-layer chromatography (TLC). The fractions containing quercetin were separated and collected using a Sephadex LH-20 column. Two different methods were employed to estimate the amount of quercetin in the extracts: a colorimetric assay developed by Christ and Müller, and a complexometric method by Belikov. The quercetin concentrations ranged between 2 mg (Tinctura Alchemilla) and 89 mg (Tinctura Hyperici) per 100 g of extract. We suggest that the mutagenicity of the 4 plant extracts is mainly due to the presence of quercetin for the following reasons: (1) all the plant extracts exhibit a mutation pattern which is very similar to that of quercetin, (2) the mutagenic potential of the extracts correlates well with their quercetin content, considering the fact that plant extracts are very complex mixtures often containing toxic or antimutagenic compounds.     

Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100

Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella typhimurium strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver S9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of S9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60% of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without S9. BP, 3MC and 3MCE were not mutagenic in either strain without S9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the S9 preparation revealed decreased AHH activity with increase in Se concentration. The decreased mutagenicity and AHH activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as Na2SO3). Inhibition of AHH activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. typhimurium TA98 and TA100.     

Measurement of drug-metabolizing systems in Salmonella typhimurium strains G46, TA1535, TA100, TA1538 and TA98

Salmonella typhimurium strains which are commonly used in the Ames test for screening potential carcinogens were examined for a number of drug-metabolizing systems. Neither cytochrome P-450 itself nor two activities catalyzed by the cytochrome P-450 system in mammalian cells, i.e., benzpyrene monooxygenase and ethoxycoumarin O-deethylation, could be detected. Nor do these bacterial strains demonstrate any ability to detoxify epoxides by hydrating them or to conjugate p-nitrophenol with glucuronic acid.On the other hand, S. typhimurium strains G46, TA1535, TA100, TA1538 and TA98 contain considerable amounts of acid-soluble thiols, approx. 5–10% of which is glutathione. These bacteria can also enzymatically conjugate glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) and can reduce oxidized glutathione using NADPH as cofactor.Thus, enzymatic and non-enzymatic reaction of immediate carcinogens with thiol groups in S. typhimurium may have a significant effect on the outcome of the Ames test in certain cases.     

Metabolic activation of 2,4-dinitrobiphenyl derivatives for their mutagenicity in Salmonella typhimurium TA98

In order to elucidate the mechanisms of mutagenic activation of nitroarenes, we tested the mutagenic potency of 18 kinds of nitroarenes including nitrated biphenyl, fluorene, phenanthrene and pyrene on Salmonella typhimurium TA98 in the absence and presence of S9 mix. The mutagenicities of 2,4-dinitrobiphenyl derivatives and 4-nitrobiphenyl were enhanced by the addition of S9. 2,4,6-Trinitrobiphenyl (3 net rev./10 micrograms without S9) was activated 60-fold by the mammalian metabolic system (181 net rev./10 micrograms with 10% S9). The mutagenic potency of 2,4,2',4'-tetranitrobiphenyl in TA98, TA98NR and TA98/1,8-DNP6 was also enhanced by the addition of 10% S9. But 1-nitropyrene and 1,3-dinitropyrene, which are well-known mutagens and carcinogens, were deactivated to 3% and 0.4%, respectively, by the addition of 10% S9. Separate addition of microsomal and cytosolic fractions slightly activated the mutagenicity of 2,4,6-trinitrobiphenyl, and 2,4,2',4'-tetranitrobiphenyl was activated not only by S9 but also by the cytosolic fraction.     

Influence of retinoids on the mutagenicity of cigarette-smoke condensate in Salmonella typhimurium TA98

The effects of 3 different retinoids (all-trans-retinol, all-trans-retinoic acid, and all-trans-retinyl acetate) on the mutagenic activity of cigarette-smoke condensate were investigated in Salmonella typhimurium TA98. Neither an enhancing nor an inhibitory effect of the retinoids on the mutagnicity of cigarette-smoke condensate was observed.     

Mutagenicity in Salmonella typhimurium TA98 and TA100 of nitroso and respective hydroxylamine compounds

Five aromatic nitroso compounds were prepared and their mutagenicity in Salmonella typhimurium strains TA98 and TA100 compared with that of the corresponding hydroxylamines and the previously studied nitroarenes. A remarkable correspondence of the dose-response curves was observed between the nitroso and the respective hydroxylamine compounds. This effect could be observed in TA98 and TA100. It was only marginally dependent on the metabolical activation by rat liver S9-mix. Even the presence of a bulky alkyl substituent either near to the functional group, or far away from it, previously shown to considerably influence the mutagenic properties of nitroarenes, does not remarkably affect the properties of the nitroso and hydroxylamine species. The similarity between the latter two is likely to be due to a fast reduction of the nitrosoarenes to the hydroxylamine species under the test conditions. It seems that enzymes are not responsible for that reduction step, because sterical crowding near the functional group does not influence that behaviour.The test results of the aromatic hydroxylamines bearing a bulky substituent show that there are at least two ways to influence the mutagenicity of an aromatic nitro compound by such a group. A substituent near the functional group (ortho-position) disturbs the enzymatic reduction of the nitro group, because 3-tert-butyl-4-hydroxylaminobiphenyl and its corresponding nitroso compound are highly mutagenic, whereas 3-tert-butyl-4-nitrobiphenyl was previously shown to be inactive even after addition of S9-mix. In contrast, 4'-tert-butyl-4-hydroxylaminobiphenyl with the tert-butyl group "far away" from the hydroxylamino functionality clearly shows decreased mutagenic activity suggesting a different influence of a substituent in that position. In addition, the substance shows only little cell toxicity even at higher concentrations. Both effects could be due to a reduced effective dose of the hydroxylamine in the cells compared to the non-alkylated compound, caused by a faster degradation of the hydroxylamine or a hindered interaction between that substance and the cells.     

A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100

'Classical nitroreductase' is an enzyme involved in the intracellular metabolic activation of mutagenic nitroarenes. The nitroreductase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1021 and YG1026) had more than 50 times higher nitrofurazone-reductase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic action of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. These results indicate that the new strains permit the efficient detection of mutagenic nitroarenes.     

Mutagenic activities of selected nitrofluoranthene derivatives in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6

The mutagenic activities of novel nitrofluoranthene derivatives in Salmonella strains TA98, TA98NR and TA98/1,8-DNP6 (with and without S9 addition) are given. These derivatives were produced from the reactions of fluoranthene (FL) and its directly mutagenic 2- and 3-nitro derivatives with covalent dinitrogen pentoxide (N2O5) in CCl4 solution at ambient temperature. The influence of the addition of a nitro group on the observed activity of the resulting di- and tri-nitrofluoranthenes is discussed.     

Mutagenicity of nitrofurans in Salmonella typhimurium TA98, TA98NR and TA98/1,8-DNP6

8 representative 2-substituted 5-nitrofurans were assayed for mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6. The tested compounds were: 5-nitro-2-furanacrylic N-(5-nitro-2-furfurylidene)hydrazide (1); furazolidone (2); 5-nitro-2-furanacrolein (3); 5-nitro-2-furaldehyde semicarbazone (4); 5-nitro-2-furaldehyde (5); nitrofurantoin (6); 5-nitro-2-furaldehyde diacetate (7); and 5-nitro-2-furoic acid (8). These compounds exhibited markedly different mutagenic activities in TA98, and these mutagenicities were similar both in the presence and the absence of rat-liver hepatic S9 activation enzymes. The mutagenic responses ranged from potent (90-300 revertants/nmole, compounds 1-3), to medium (about 10 revertants/nmole, compounds 4 and 6), to weak (0-4 revertants/nmole, compounds 5, 7 and 8). The mutagenicity of 3 was similar in all 3 tester strains, while compound 8 was essentially inactive. The mutagenicities of 1, 4, 5 and 7 were decreased 30-75% in TA98NR, while 2 and 6 showed an even greater depression of activity in this strain. Compound 6 with S9 was about equally mutagenic in TA98 and TA98/1,8-DNP6, while the activities of 6 without S9 and 2 and 7 both with and without S9 were 50-75% lower in TA98/1,8-DNP6. Compounds 1, 4 and 5 were only about 5-10% as mutagenic in TA98/1,8-DNP6 as in TA98. These results suggest that: (i) nitrofurans and their S9-mediated metabolites have similar mutagenic potencies; (ii) with the possible exception of No. 3, nitroreduction is the major route of mutagenic activation for these nitrofurans; and (iii) for compounds 2, 6 and 7, both the presumed N-hydroxy and N,O-ester derivatives of the corresponding aminofuran metabolites appear to lead to mutations.     

Genetic toxicology evaluation of commercial beers. II. Mutagenic activity of commercial beer products in Salmonella typhimurium strains TA98, TA100 and TA102

5 concentrated extracts of commercial beers were prepared using XAD-2 resin. The residues were subjected to evaluation for mutagenic activity in Salmonella typhimurium strains TA98, TA100 and TA102. The tests were conducted using preincubation protocols including provisions for S9 metabolic activation. Although the extracts did produce moderate toxicity to the Salmonella organisms used in the assays, none of the residues were found to induce mutation up to their maximum testable concentrations.     

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.     

Mutagenicity of coal-pyrolyzed products and their photochemical reaction products with nitrogen oxides in Salmonella typhimurium TA98 and TA100

The chemical class separation of coal-pyrolyzed products and the photochemical reaction of these fractions with nitrogen oxides in the experimental chamber, and the application of a short-term mutagenicity test were investigated. The altered products from the fraction hydroxy polycyclic aromatic compounds in a simulated atmosphere containing a small volume of nitrogen oxides under irradiation with a xenon lamp were the most potent mutagenic fraction among all the fractions tested against Salmonella typhimurium, both TA98 and TA100, with or without S9.