Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: II. With exogenous activation

In order to determine the usefulness of selected chemicals as potential reference materials for calibrating the Salmonella assay, two laboratories tested a series of Salmonella mutagens that require exogenous activation. When the variance for individual substances within a bioassay is sufficiently low and the rankings of those substances are of acceptable consistency, they can later be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The purpose of this project, therefore, was to evaluate the variability in the mutagenic response of potential reference chemicals that require exogenous metabolic activation in the standard plate-incorporation Salmonella mutagenicity assay, and to develop ranking criteria for mutagenic activity based on these data. Ten indirect-acting mutagens were tested in two laboratories using Salmonella typhimurium TA100 and an Aroclor-induced rat liver S9. Each laboratory conducted four definitive testing rounds. A different batch of S9 was utilized for every two rounds. Of the 10 chemicals tested only 2-anthramine had a mean slope value greater than 1000 revertants/micrograms. Three chemicals had slope values between 1000 and 100; and five chemicals had slope values between 100 and 10. The remaining compound, 9,10-dimethyl-1,2-benz[a]anthracene, could not be placed into a single category because it had slope values on either side of 100 revertants per mg. Coefficients of variance were low (i.e., below 25% in most cases). The low variability achieved in this study may be accounted for by two parameters of the study. First, based on Claxton et al. (1991a) and the S9 optimization for three compounds, the amount of S9 was calibrated to a set amount of protein per plate (1.1 mg/plate). Secondly, the 10 test doses were placed in the initial, linear, nontoxic portion of the dose-response curves. The use of ten closely spaced, nontoxic doses allowed for a more accurate estimate of the slope.     

Detection of mutagens produced by fungi with the Salmonella typhimurium assay.

Forty-one fungal isolates (one isolate per species) representing common plant pathogens and food crop contaminants were grown on sterile, polished rice and assayed for mutagenic activity in the Salmonella typhimurium-microsome system. Initially, single doses of aqueous and chloroform extracts of the moldy rice were assayed against the TA100 tester strain by incorporating extracts into the growth medium and by applying small quantities on disks placed on the agar surface. Suspected activity was examined further by analysis of several doses in the plate incorporation assay. Extracts of two aflatoxin-producing isolates (Aspergillus flavus and A. parasiticus) showed pronounced mutagenic activity, as did extracts of five other isolates (A. heterothallicus, A. nidulans, A. terricola, Alternaria tenuis, and Fusarium moniliforme) which did not contain detectable aflatoxins. Seven additional isolates (Botrytis cineria, Ceratocystis fimbriata, Cladosporium herbarum, Fusarium solani f. sp. pisi, Penicillium oxalicum, Thermomyces lanuginosus, and Verticilium albo-atrum) revealed activity which was possibly mutagenic; i.e., mutagenic responses were not observed in both the disk and incorporation assays, and clear dose-related activity was not observed in the incorporation assay. Extracts of the remaining fungi were not mutagenic in the bacterial assay.     

Detection of mutagens produced by fungi with the Salmonella typhimurium assay.

Forty-one fungal isolates (one isolate per species) representing common plant pathogens and food crop contaminants were grown on sterile, polished rice and assayed for mutagenic activity in the Salmonella typhimurium-microsome system. Initially, single doses of aqueous and chloroform extracts of the moldy rice were assayed against the TA100 tester strain by incorporating extracts into the growth medium and by applying small quantities on disks placed on the agar surface. Suspected activity was examined further by analysis of several doses in the plate incorporation assay. Extracts of two aflatoxin-producing isolates (Aspergillus flavus and A. parasiticus) showed pronounced mutagenic activity, as did extracts of five other isolates (A. heterothallicus, A. nidulans, A. terricola, Alternaria tenuis, and Fusarium moniliforme) which did not contain detectable aflatoxins. Seven additional isolates (Botrytis cineria, Ceratocystis fimbriata, Cladosporium herbarum, Fusarium solani f. sp. pisi, Penicillium oxalicum, Thermomyces lanuginosus, and Verticilium albo-atrum) revealed activity which was possibly mutagenic; i.e., mutagenic responses were not observed in both the disk and incorporation assays, and clear dose-related activity was not observed in the incorporation assay. Extracts of the remaining fungi were not mutagenic in the bacterial assay.     

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.     

Forward mutations to arabinose resistance in Salmonella typhimurium strains: a sensitive assay for mutagenicity testing.

The forward-mutation assay using the L-arabinose-sensitive strain SV3 of Salmonella typhimurium has been calibrated against a selected set of mutagens. Strain SV3 is sensitive to chemicals causing base-pair substitutions, frameshift mutations and deletions. New strains deficient for the excision-repair system or the lipopolysaccharide barrier or both have been selected from strain SV3. The additional mutations do not affect the independence of the assay from experimental artifacts due to physiological or lethal damage or differences in plating density. The new strains are more sensitive than SV3 to certain mutagens. Techniques for using this set of strains are presented and their relative advantages discussed.     

Lack of mutagenicity to Salmonella typhimurium of some Fusarium mycotoxins.

The mutagenicity of eight Fusarium toxins (mono-, di-, and triacetoxyscirpenol, T-2 toxin, deoxynivalenol, 3-acetyl-deoxynivalenol, zearalenone, and moniliformin) and of two positive controls (aflatoxin B1 and sterigmatocystin) to histidine-requiring strains TA 98, 100, 1535, and 1537 of Salmonella typhimurium was tested both with and without metabolic activation. Both aflatoxin B1 and sterigmatocystin, but none of the eight Fusarium toxins, were mutagenic to S. typhimurium. The lack of mutagenic activity of T-2 toxin and diacetoxyscirpenol supports the negative results that have been obtained with in vivo carcinogenicity tests. The negative mutagenicity of the four other 12,13-epoxytrichothecenes tested, and of zearalenone and moniliformin, could not be correlated with in vivo tests because published accounts of their chronic toxicity were not available.     

Lack of mutagenicity to Salmonella typhimurium of some Fusarium mycotoxins.

The mutagenicity of eight Fusarium toxins (mono-, di-, and triacetoxyscirpenol, T-2 toxin, deoxynivalenol, 3-acetyl-deoxynivalenol, zearalenone, and moniliformin) and of two positive controls (aflatoxin B1 and sterigmatocystin) to histidine-requiring strains TA 98, 100, 1535, and 1537 of Salmonella typhimurium was tested both with and without metabolic activation. Both aflatoxin B1 and sterigmatocystin, but none of the eight Fusarium toxins, were mutagenic to S. typhimurium. The lack of mutagenic activity of T-2 toxin and diacetoxyscirpenol supports the negative results that have been obtained with in vivo carcinogenicity tests. The negative mutagenicity of the four other 12,13-epoxytrichothecenes tested, and of zearalenone and moniliformin, could not be correlated with in vivo tests because published accounts of their chronic toxicity were not available.     

Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.     

The use of the Salmonella/microsomal assay to determine mutagenicity in paired chemical mixtures.

The mutagenicities of two sets of chemicals acting singly and in pairwise combinations were determined by use of the Salmonella/microsomal assay. The first set consisted of the promutagens of benzo(a)pyrene and benzo(rst)pentaphene. The second set contained the direct-acting mutagens methyl-nitro-nitroso-guanidine and ethyl methane sulfonate. In the tests with the promutagens, the quantities of S-9 mix were varied over the range of 0.05 ml to 1.0 ml with increasing quantities of each chemical. The mutagenic responses or production of revertant colonies of the promutagens, acting singly and in pairwise combinations failed to show an additive effect. Excess quantities of S-9 mix appeared to inhibit partially or totally the mutagenic activity of each chemical, although for each particular dose there was an optimal quantity of S-9 mix to induce maximum activity. However, the direct-acting mutagens produced, individually, almost linear dose responses with increasing concentrations. In pairwise combinations, these chemicals also showed linear responses that closely approximated the theoretical additivity indicating that the mutagenicity of the mixtures was the sum of the activities of each component.     

Toxicity and mutagenicity of hexavalent chromium on Salmonella typhimurium.

Four hexavalent and two trivalent chromium compounds were tested for toxicity and mutagenicity by means of the Salmonella typhimurium/mammalian-microsome test. All hexavalent compounds yielded a complete inhibition of bacterial growth at doses of 400 to 800 mug/plate, a significant increase of his(+) revertant colonies at doses ranging from 10 to 200 mug, and no effect at doses of less than 10 mug. The distinctive sensitivity of the four Salmonella strains tested (TA1535, TA1537, TA98, and TA100) suggested that hexavalent chromium directly interacts with bacterial deoxyribonucleic acid by causing both frameshift mutations and basepair substitutions. The latter mutations, which are prevalent, are amplified by an error-prone recombinational repair of the damaged deoxyribonucleic acid. On the average, 1 mumol of hexavalent chromium yielded approximately 500 revertants of the TA100 strain, irrespective of the compound tested (sodium dichromate, calcium chromate, potassium chromate, or chromic acid). The mutagenic potency of the hexavalent metal was not enhanced by adding the microsomal fraction of rat hepatocytes, induced either with sodium barbital or with Aroclor 1254. The two trivalent compounds (chromium potassium sulfate and chromic chloride), with or without the microsomal fraction, were neither toxic nor mutagenic for the bacterial tester strains.     

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.     

Hydrazines as mutagens in a histidine-requiring auxotroph of Salmonella typhimurium.

Hydrazines have been found naturally in tobacco and mushrooms. Other hydrazines are used in industry, medicine, and agriculture. Although about 38 hydrazines are carcinogenic, few, if any, have been tested successfully in rapid bacterial mutagenesis assays. We have utilized a tester strain of Salmonella typhimurium (TA1530) in order to determine the mutagenic activity of a number of hydrazines and related compounds. This strain is thus shown to be effective as a tester organism for the facile detection of hydrazines as mutagens.     

Diethylstilbestrol and 11 derivatives: a mutagenicity study with Salmonella typhimurium.

Diethylstilbestrol was tested for mutagenicity with his- S. typhimurium strains under 10 different matabolic situations (no exogenous metabolizing system; S9 mix from liver homogenate of rats induced with Aroclor 1254, with or without inhibition of epoxide hydratase; liver and/or kidney S9 mix from control or hamsters treated with Aroclor 1254; horse-radish peroxidase + H2O2). Under none of these conditions did diethylstilbestrol give any indication of a mutagenic effect. Furthermore, 11 metabolites and other derivatives of diethylstilbestrol, 2 of them potent inducers of sister-chromatid exchange in cultured fibroblasts, were not mutagenic with any of the 4 tester strains (S. typhimurium TA100, TA98, TA1537, TA1535) in the presence or absence of S9 mix from liver homogenate of rats induced with Aroclor 1254. Thus, one of the few known human carcinogens is very resistant to detection by the mammalian enzyme-mediated Salmonella typhimurium mutagenicity test (Ames test). This is especially remarkable since the metabolizing systems used included: (1) some of very high metabolic activity (S9 mix from liver homogenate of rats and hamsters induced with Aroclor 1254); (2) metabolizing systems from organs susceptible to the carcinogenic activity of diethylstilbestrol (hamster kidney); as well as (3) a mixture of (1) and (2) in case both activities are required for the carcinogenic effect in the whole animal.     

In vitro metabolic activation of chemical mutagens. I. Development of an in vitro mutagenicity assay liver microsomal enzymes for the activation of dimethylnitrosamine to a mutagen

Qualitative and quantitative assay were developed to study the in vitro enzymatic activation of dimethylnitrosamine (DMNA) to its mutagenic form. Three different fractions from mouse liver homogenates, including purified microsomes, were employed for the activation, and several parameters of the assay were investigated. Qualitative tests were conducted to measure the ability of hepatic enzymes obtained from six mammalian specie to activate DMNA. A comparison between two inbred mouse strains using the in vitro activation assay demonstrated that this technique might be a useful tool in quantitatively measuring differences in genetically influenced levels of DMNA metabolism in individual animals and their tissues.     

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.     

Studies on mutagenicity of irradiated sugar solutions in Salmonella typhimurium.

Irradiated sugar solutions are mutagenic towards Salmonella typhimurium, the effect being dose-dependent up to 2.0 Mrad. At all doses, ribose solution exhibited greater mutagenicity than did sucrose solution. The mutagenic effect was observed only in dividing cells and appears to be directly related to the growth rate. A larger proportion of revertants was observed after incubation with irradiated sugar solution for a period of 4 h than for 24 h. Irradiation of the sugar solutions in the frozen conditions was effective in completely preventing the development of mutagenic potential. Post-irradiation storage of the sugar solutions for a prolonged period (25 weeks) also minimized their mutagenic effect. The irradiated sugar solutions gave rise to both missense and frame-shift (additon as well as deletion) types of mutation; ribose was more effective in inducing the latter type. The irradiated sugar solutions failed to show a mutagenic response in the host-mediated assay with mice as the mammalian host.