Mutagenic evaluation of nitroparaffins in the salmonella typhimurium/mammalian-microsome test and the micronucleus test

Three nitroparaffins (nitroethane, 1-nitropropane, and 2-nitropropane) were studied in the Salmonella typhimurium/mammalian microsome (Ames) test, with and without microsomal activation systems. Nitroethane and 2-nitropropane also were studied in an in vivo mutagenic (micronucleus) test. These studies were undertaken because these solvents are widely used in the chemical and pharmaceutical industries and 2-nitropropane was reported to cause liver cancer in rats exposed by the inhalation route. Neither nitroethane nor 1-nitropropane was active in the Ames test with Salmonella tester-strains TA1537, TA92, TA98, or TA100. However, 2-nitropropane produced a significant increase in revertants in all of these tester strains, particularly strain TA100, where 3μl/plate doubled the number of revertants in the presence of microsomal enzymes. Negative results were obtained with both nitroethane and 2-nitropropane in micronucleus tests. These studies have shown that 2-nitropropane has the potential for causing point mutations in a microbial test system. However, this compound probably will not cause a chromosome mutation of the clastogenic type.     

Evaluation of the mutagenicity of an N-nitroso contaminant of the sunscreen Padimate O: N-nitroso-N-methyl-p-aminobenzoic acid, 2-ethylhexyl ester (NPABAO).

The nitrosamine contaminant, N-nitroso-N-methyl-p-aminobenzoic acid, 2-ethylhexyl ester (NPABAO), of the major sunscreen ingredient Padimate O (4-N,N'-dimethylamino-benzoic acid, 2-ethylhexyl ester) was synthesized and tested for mutagenicity in the Salmonella typhimurium and mouse lymphoma L5178Y TK +/- assays. In contrast to the previously reported positive responses in S. typhimurium tester strains TA100 and TA1535 [Loeppky et al., 1991], there were no increases in the number of revertants with strains TA98, TA100, TA1535, and TA1538 in either the Salmonella plate incorporation [Ames et al., 1975] or preincubation [Yahagi et al., 1977] assays. Additional testing with Salmonella, following the modified preincubation procedure [Rogan, 1990] that gave the initial positive response, was also negative. Data from the mouse lymphoma assays were also uniformly negative. During synthesis of NPABAO, small amounts of 4-N,N'-dimethylamino-3-nitrobenzoic acid, 2-ethylhexyl ester (DMANBAO) can be formed. To determine whether the reported positive mutagenicity response of NPABAO could be the result of trace amounts of DMANBAO in the NPABAO, that compound was also synthesized and tested for mutagenicity with Salmonella. Positive responses were obtained with tester strains TA98 and TA 1538 but not with TA100 and TA1535, indicating that DMANBAO was not responsible for the increase in revertants originally reported.     

A protocol for the combined biochemical and serological identification of the Ames mutagen tester strains as Salmonella typhimurium

Previously published reports have noted biochemical reactions atypical of Salmonella among the Ames tester strains of Salmonella typhimurium, and an inability to assign the strains to a specific Salmonella O (heat-stable cell wall) antigen group. We studied the biochemistry and serology of strains TA97, 98, 100, 102, 104, 1535, 1537, and 1538 in an attempt to develop a protocol to correctly speciate the strains. Biochemical reactions of all eight strains using standard media supplemented with histidine and biotin were consistent with those of the genus Salmonella. Strains TA100, 104, and 1535 were assigned to Salmonella O groups using bacteria treated with hot ethanol (White schema). H (flagellar) antigen assignments were performed successfully with seven of the eight strains. Two H antigen assignments required the use of the Craigie tube test for selection of motile revertants. Combining our biochemical and serological results obtained by this protocol, we were able to correctly speciate TA100, 104, and 1535 as Salmonella typhimurium. Our results demonstrate that representatives of the tester strains can be correctly speciated provided that procedures are followed that allow for the unusual nutrient requirements, the deep rough cell wall mutation, and the variably deficient motility of these organisms.     

Influence of the triazine ring on the mutagenicity of triazinoindoles and some congeners.

Three compounds, which could be considered as precursors or derivatives of the 3-(4'-substituted-benzylidenamino)5H- 1,2,3-triazin[5,4b]indol-4-one series, were selected from the study of their mutagenic activity. Ames tests were performed study of their mutagenic activity. Ames tests were performed using the Salmonella typhimurium strains TA97, TA98, TA100, and TA102, according to the preincubation procedure, both with and without metabolic activation. The 3-amino-5H-1,2,3-triazin[5,4b]indol-4-one has been shown to be a strong S9-independent mutagen, which reverts frameshift and substitution mutations. Nevertheless its potency increases with the addition of microsomal fraction. In contrast, the 2-benzyliden-1-(3-aminoindol)-2-carbohydrazide and the 3-aminoindol-2-carbohydrazide congeners were not mutagenic. These results suggest that the 1,2,3-triazine ring is the principle substructure responsible for the mutagenicity of the triazinoindole congeners studied.     

Lack of genotoxicity of silver iodide in the SCE assay in vitro, in vivo, and in the Ames/microsome test

Silver iodide was evaluated for mutagenicity in the Ames/microsome test (strains TA 1535, TA 102, TA 97, and TA 98) and for the ability to induce Sister Chromatid Exchanges (SCE) in human cultured lymphocytes and in P388 lymphocytic leukemia cells cultured in the mouse peritoneal cavity. From the cytogenetic in vitro studies, it was observed that silver iodide, either in acetone solutions or as a suspension with polyacrilamide, scarcely causes a doubling effect on SCEs at nearly toxic concentrations (1 microg/ml). Such a doubling effect by silver iodide on SCEs in P388 leukemia cells in vivo was not achieved even after using 100 microg/g mouse body weight. In the Ames/microsome test actually a doubling effect on revertants was only isolately achieved with 30 microg/ml in TA 102 (S9-) and at 150 microg/ml in TA 97 (S9+) doses, which appear to be nearly toxic for bacteria.     

The mutagenic and SOS-inducing potential of the soluble organic fraction collected from diesel particulate emissions

Studies involving the Ames Salmonella mutagenicity test and the Bacillus subtilis comptest have demonstrated that the soluble organic fraction of diesel particulate is potentially mutagenic and DNA damaging. The soluble organic fraction was extracted from exhaust particulate samples collected from four different diesel engines operated at specified conditions. For each fraction collected, an increase in the concentration of the organic material resulted in a subsequent increase in the number of histidine prototrophs obtained when this material was added to the histidine auxotrophic strains that comprise the Ames Salmonella test. Specifically, the number of induced revertants, for strains TA98 and TA100, ranged from less than one revertant per microgram of sample to 29 revertants per microgram of sample. The ability of these organic fractions to induce bacterial SOS functions also was determined by exposing competent cultures of Bacillus subtilis strain RUB827 to increasing concentrations of these extracts. With varying efficiencies, these samples were positive in their ability to induce the SOS system of B subtilis. Significantly, the toxicity of these mutagenic and DNA damaging samples never resulted in more than 95% killing, even for the highest concentrations tested in the Salmonella and B subtilis assay.     

Genotoxicity of 2-halocinnamaldehydes in two bacterial assays. Induction of SOS repair and frame-shift mutation

2-Chlorocinnamaldehyde and 2-bromocinnamaldehyde, compoundsof practical interest, for example, as bacterioddes and fungicidesor for utilization in light sensitive layers, were tested inthe Ames preincubation test with various Salmonella typhimuriumstrains, and in the SOS chromotest with Escherichia coli PQ37. 2-Chlorocinnamaldehyde was clearly mutagenic in strain TA100 (6081 revertants/µmol) and in strain TA 98 (3050 revertants/µmol)without S9 mix, and was clearly positive in the SOS chromotest(SOSIP = 0.181). 2-Bromocinnamaldehyde was a strong mutagenin strain TA 100 (105, 500 revertants/µmol), in strainTA98 (41567 revertants/µmol) and in strain TA 1538 (15825revertants/ µmol), and also unambiguously mutagenic instrain TA 1535 (2110 revertants/µmol) without S9 mix.The SOSIP in the SOS chromotest was 1.5. Addition of S9 mixled to a marked decrease in the mutagenic activity of 2-bromocinnamaldehydein all strains tested. In the case of strain TA 1535, mutagenicactivity was abolished or not significant in the presence ofS9 mix. The possible primary mechanisms underlying these mutageniceffects are discussed. Frame-shift activity of these halocinnamaldehydescan be explained by their planar structure. ¹To whom correspondence should be addressed     

Synthesis,chemical characterization,and mutagenic activities of promutagens produced by pyrolysis of proteinaceous substances

Chemical and mutagenic characteristics of TRP-P-1, TRP-P-2, GLU-P-1, GLU-P-2, GLOB-P-1, and GLOB-P-2 were evaluated. We synthesized TRP-P-1 and TRP-P-2 and also obtained samples of these compounds from a commercial source. By GC-MS analysis, the samples of TRP-P-2, GLUs, and GLOBs were more than 99% pure; but the samples of TRP-P-1 contained 11–17% of different impurities. These impurities had no effect on the mutagenicity of these chemicals in the Ames test using either strain TA98 or TA100 of Salmonella typhimurium. All six compounds were inactive without metabolic activation and the γ-carbolines (TRPs) were more active in both strains than the α-carbolines (GLOBs). GLU-P-1 was the most active of the promutagens tested in both TA98 (41,000 revertants/μg) and TA100 (6,580 revertants (μg). In TA98 the order of activity was GLU-P-1 > TRP-P-2 > TRP-P-1 > GLU-P-2 ? GLOB-P-2 > GLOB-P-1. In TA100 the order of activity was GLU-P-1 ? GLU-P-2 > TRP-P-2 ? TRP-P-1 ? GLOB-P-1 ? GLOB-P-2. We determined the UV absorption and fluorescence characteristics of these compounds, and of HM and NHM, and established an HPLC procedure for their resolution. Sensitivities in the picogram range were attainable using fluorescence detection.     

Evaluation of the mutagenic and genotoxic activities of 48 nitroimidazoles and related imidazole derivatives by the Ames test and the SOS chromotest.

The mutagenic and genotoxic activities of 48 nitroimidazoles and related imidazole derivatives have been evaluated by using modified versions of the Ames test and the SOS Chromotest. Salmonella typhimurium tester strain TA 100 was used with and without metabolic activation in the Ames test and Escherichia coli tester Strain PQ 37 was used with and without metabolic activation in the SOS Chromotest. Including metronidazole and dimetridazole, 45 derivatives were mutagenic and genotoxic. The mutagenic potencies (MP) ranged from 0.127 to 53,717 revertants/nmol while the SOS induction powers (SOSIP) ranged from 0.00131 to 107 IF/nmol. The overall correlation between MP and SOSIP was r = 0.845 (n = 84) as calculated by linear regression analysis. A higher correlation was observed between MP and SOSIP without the S9 mix than with it. Among the imidazole derivatives, the 5-nitroimidazoles with a lactam ring at the 2-position showed the highest MP and SOSIP. The presence of a nitro group at the 5-position was critical for the mutagenicity and the genotoxicity of the derivatives. Substituents at the 1- and 2-positions were also found to modulate these activities.     

Metabolic formation, synthesis and genotoxicity of the N-hydroxy derivative of the food mutagen 2-amino-1-methyl-6-phenylimidazo (4,5-b) pyridine (PhIP)

Hepatic microsomes from rats pretreated with PCB were found to metabolize the food mutagen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) to two major metabolites, one of which was identified as the N-hydroxy derivative, 2-hydroxy-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-OH-PhIP). This identification was based on mass spectral (MS), UV and HPLC data by comparison with N-OH-PhIP prepared by chemical synthesis, as well as the specific activity of the compound in the Ames Salmonella test. Synthetic N-OH-PhIP was prepared by catalytic reduction of the nitro derivative of PhIP, which was synthesized from PhIP by diazotization and reaction with sodium nitrite. N-OH-PhIP was mutagenic to Salmonella typhimurium TA98 without metabolic activation and had a specific mutagenic activity of 2700 revertants/nmol. N-OH-PhIP thus seems to be a proximate mutagenic metabolite of PhIP. Other direct acting mutagens were not detected in the microsomal incubation mixture after HPLC separation. N-OH-PhIP also induced sister chromatid exchange (SCE) in Chinese hamster ovary cells (CHO cells) without metabolic activation. The specific activity of N-OH-PhIP in this assay was approximately 3 times higher than the activity of PhIP with microsomal activation.     

Ames试验在羟基磷灰石人工骨材料评价中的应用

目的通过鼠伤寒沙门氏菌回复突变试验(Ames试验)探讨羟基磷灰石人工骨材料的遗传毒性特征,以评价该材料的潜在致突变性。方法选择生理盐水(SC)和二甲基亚砜(DMSO)两种溶剂对试验样品浸提,采用平板掺入法,计数TA97、TA98、TA100和TA102菌株在活化和非活化条件下的回变菌落数,以检测其致突变比值。结果试验阴性对照和阳性对照结果有效,试验成立。采用SC或DMSO浸提,浸提原液均未引起试验菌株回变菌落数超过阴性对照的2倍,即致突变比值MR均<2。结论在本实验条件下,试验样品SC浸提原液及DMSO浸提原液对试验菌株无诱变性。     

Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. IV. Relationship between the number of his- bacteria plated and number of his+ revertants scored in the Salmonella mutagenicity test

Five laboratories participated in a joint ring study to investigate the role of bacterial cell number in the Salmonella mutagenicity test. A strictly standardized protocol, using sodium azide and TA 1535, was developed and employed to test the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with different dilutions of Salmonella typhimurium TA-100 and TA-1535 cultures. All laboratories detected the mutagenic activity of sodium azide with only a 2-fold variation of test results. For MNNG the interlaboratory variation was approximately 5-fold. Decreasing numbers of test bacteria employed resulted in lower numbers of MNNG-induced revertants in all laboratories. The number of preexisting revertants decreased in direct proportion to the reduced cell content, whereas the number of spontaneous revertants was not as greatly affected. A critical amount of test bacteria was required in order to obtain numbers of induced revertants which were equal to twice the number of spontaneous revertants. Two evaluation parameters which may be employed to describe the mutagenicity of a compound are compared.     

Mutation spectrum in Salmonella induced by environmental tobacco smoke.

Environmental tobacco smoke (ETS) is a major source of indoor air pollution. Extractable-respirable particulate (ERP) from the ETS-contaminated indoor air (ERP-ETS) was collected from six passenger train cars and one control room. The mutagenicity of ERP-ETS was tested in the Ames/Salmonella test in the presence of male rat liver microsomal fraction S9. The mutation spectrum of ERP-ETS was determined by colony probe hybridization and polymerase chain reaction/DNA sequence analysis in approximately 2,370 His+ revertants. The results indicate that the majority of ERP-ETS-induced mutations were a two-base deletion of GC or CG within the hotspot sequence of CGCGCGCG at the frameshift hisD3052 allele in strain TA98. The ERP-ETS from the control room induced approximately 94.3% such deletions, while the ERP-ETS collected from the passenger cars induced approximately 89.6% such deletions. The ERP-ETS either from the control room or from the passenger cars induced approximately 74% C/G --> A/T transversions, and approximately 23% C/G --> T/A transitions within the primary target CCC at the hisG46 allele in strain TA100.     

Mutagenicity of lichen constituents

Usnic acid (the most abundant lichen constituents), physodic, and physodalic acids isolated from Hypogymnia enteromorph (Ach.) Nyl. were tested for mutagenicity in the Ames Salmonella/microsome assay. Physodalic acid exhibited clear dose-related mutagenicity against Salmonella typhimurium strain TA 100 with or without S9 mix in both plate-incorporation and preincubation assays. The addition of S9 mix increased the number of revertants approximately threefold and fourfold in preincubation and plate-incorporation assays, respectively.     

Comparison of the Salmonella (Ames) test, umu tests, and the SOS Chromotests for detecting genotoxins

The limits of detection of 10 genotoxins representing 7 chemical classes with varying structures and modes of action were compared using the Ames test (Salmonella plate-incorporation test) with 2 tester strains, 2 standard colorimetric methods (the umu test and SOS Chromotest), and modifications of the umu and SOS Chromotests developed during the course of this study. The purpose of the study was to determine the sensitivity and reproducibility of each of the six methods. The sensitivities of the methods were compared using two criteria: the concentrations required for doubling responses, and the minimum concentrations required to produce statistically significant increases from background controls. The Ames test with strains TA98 and TA100 was ranked as the most sensitive method more often than the others, but the results indicated that the umu tests were statistically equivalent to the Ames test. The original SOS Chromotest kit method was highly sensitive in detecting the direct acting genotoxins, but neither SOS test was as sensitive as the other methods in detecting indirect acting genotoxins. The umu microtiter plate test is the least expensive of the assays and would be the most suitable for screening large numbers of environmental samples.     

Mutagenic activity of epoxy embedding reagents employed in electron microscopy

Components of epoxy-based embedding media used in electron microscopy were examined for their mutagenicity in the Ames Salmonella system. The compounds, singly and in combination, were shown to be active with strain TA100 (base substitution indicator) but not with TA98 (frameshift indicator). When tested separately the epoxy resins Araldite, Epon, and vinyl cyclohexen dioxide (VCD) and the plasticizer diglycidyl ether of propylene glycol (DER-736) were found to be significantly mutagenic. These active compounds, in combination with liver mixed oxidase preparation (S9), showed increased mutagenicity over similar preparations in the absence of microsomal activation.     

Mutagenic activity of epoxy embedding reagents employed in electron microscopy.

Components of epoxy-based embedding media used in electron microscopy were examined for their mutagenicity in the Ames Salmonella system. The compounds, singly and in combination, were shown to be active with strain TA100 (base substitution indicator) but not with TA98 (frameshift indicator). When tested separately the epoxy resins Araldite, Epon, and vinyl cyclohexen dioxide (VCD) and the plasticizer diglycidyl ether of propylene glycol (DER-736) were found to be significantly mutagenic. These active compounds, in combination with liver mixed oxidase preparation (S9), showed increased mutagenicity over similar preparations in the absence of microsomal activation.     

Genotoxidty of 2-nitropropane and 1-nitropropane in Salmonella typhimurium and human lymphocytes

A 10- and 12-fold increase of revertant numbers could be demonstratedfor 2-nitropropane (2-NP of >99% purity) tested in the preincubationassay with Salmonella typhimurium strains TA 100 and TA 98 inthe presence and absence of s9 mix. In the nitroreductase-deficientstrains TA 100NR and TA 98NR, 2-NP was less mutagenic than inthe parent strains. In human lymphocytes the induction of aweak clastogenic effect and of sister chromatid exchanges requiredexogenous metabolic activation. No significant mutagenic orcytogenetic response was found with 1-nitropropane of 97% purityin S.typhimurium or human lymphocytes.     

In vitro genotoxicity of dyes present in colored smoke munitions

Genetic toxicology studies were conducted on organic dyes and mixtures used in colored smoke munitions. The dyes studied included Solvent Red 1; two different batches (Lot 1 and Lot 2) of Disperse Red 11; terephthalic acid; and a mixture of 25 parts Solvent Red 1, 5 parts Disperse Red 11, and 16 parts terephthalic acid. The dyes were evaluated for their ability to produce mutations in Salmonella bacterial strains and in Chinese hamster ovary (CHO) cells. The dyes were also tested in CHO cells to determine cytotoxicity and the induction of sister chromatid exchanges and chromosome aberration. None of the dyes were genotoxic in the standard Ames assay using bacterial strain TA1535 or TA100 with or without the addition of S-9 or in TA98 and TA1538 without S-9. With S-9, Disperse Red 11 (Lot 2) showed significant mutagenic activity in TA98 and TA1538 which increased as a function of S-9 concentration. However, the maximum level of mutagenic activity detected was low (3.8 revertants/micrograms). The azo dye Solvent Red 1 was also negative in a pre-incubation assay designed to reduce azo compounds to free amines. Solvent Red 1 was cytotoxic to mammalian cells, caused a significant increase in SCE, but was not mutagenic or clastogenic. Disperse Red 11 (Lot 1 and Lot 2) were not cytotoxic or clastogenic but produced an increase in cell cycle time and SCE frequency. Only Disperse Red 11 (Lot 2) increased mutations in the CHO/hypoxanthine-guanine phosphoribosyltransferase (HGPRT) assay. The mutagenic activity of the dye mixture was not significant, suggesting no synergistic interaction between the dyes. These studies demonstrated that none of the dyes was clastogenic and that a contaminant in Disperse Red 11 (Lot 2) may be responsible for the weak mutagenic activity in both mammalian and bacterial cell systems.     

Mutagenic potentials of dental cements as detected by the Salmonella/microsome test

The potential mutagenicity of a zinc phosphate (Poscal), a polycarboxylate (Aqualox) and glass ionomer cements with (Argion) and without (Meron) silver reinforcement were characterized by employing the Ames Salmonella/microsome test. The materials were eluted in dimethyl sulphoxide or physiologic saline and the aliquots were used either immediately or after an incubation period of 24h at 37 degrees C. Mutagenic effects of the materials were tested on Salmonella typhimurium strains TA 98, TA 100, TA 102 and TA 1535 using the standard plate incorporation assay, and in the presence or absence of S9 fraction from rat liver. Poscal and Aqualox elicited mutagenic effects on S. typhimurium TA 98 and TA 1535, whereas Meron exhibited mutagenic effects on S. typhimurium TA 98. No mutagenic effects were detected for Argion. The type of solvent, dose of the material and incubation as well as the interactions between these factors exhibited varying degrees of influences on the mutagenic activities of the cements (P<0.05 and P<0.1). We conclude that zinc phosphate, polycarboxylate, and glass ionomer cements may have possible mutagenic activities.