Number Of Spontaneous Revertants Research Articles

New possibilities of the Ames test for evaluation of mutagenicity of technical products of active ingredients of pesticides

Introduction. The Ames test is the one of the most popular methods for mutagenicity evaluation of environmental factors. In some cases, this method is suggested to be the only and sufficient assay for the first stage of the equivalence assessment of pesticide technical grade active ingredients (TGAI) to the original products. A limitation of the Ames test is related to the impossibility of an objective equivalence assessment of some cytotoxic TGAIs, in particular, sulfonylureas, and triazolpyrimidines. Based on the mode of action of the pesticides belongs to these chemical classes, we suggested a modification of the plate incorporation method protocol of the Ames test to the increase of maximal non-cytotoxic concentration up to the 5 mg/plate recommended by regulatory documents. Materials and methods. The five strains of Salmonella typhimurium TA98, TA100, TA1535, TA97, TA102 were used. The modification of the protocol included a supplementation of the top agar with isoleucine (1-5 mM). Results. The maximum non-cytotoxic concentrations of thifensulfuron-methyl and florasulam using the standard top agar did not exceed 0.05-0.125 mg/plate. The enrichment of the top agar with isoleucine allowed evaluating the mutagenicity of the substances up to the maximal recommended concentration of 5.0 mg/plate. The number of spontaneous revertants was within the historical limits of the laboratory control obtained under standard conditions. Positive controls showed pronounced mutagenic effects in case of all strains with and without metabolic activation (p≤0.05). Limitations. Mutagenicity was evaluated only for TGAIs, which are acetohydroxyacid synthase inhibitors. Conclusion. The application of the modified Ames test protocol for mutagenicity assessment of TGAIs from the classes of sulfonylureas and triazolpyrimidines under supplementation of the top agar with isoleucine is a more objective way to evaluate their mutagenicity. The proposed protocol expands the possibilities of revealing dangerous mutagenic impurities that may occur in TGAIs in the small quantities, and after entering the environment can cause the gain in the mutation level in living organisms.

Assessment of experimental conditions affecting spontaneous mutation level of Salmonella strains used in the Ames test

Introduction. The bacterial reverse gene mutations test (the Ames test) is widely used to assess chemicals’ mutagenic activity. The spontaneous mutation level of test strains is a mandatory characteristic that has to be monitored in a laboratory performing mutagenicity studies using the Ames test. In this regard, it is important to assess the factors affecting the spontaneous mutation level in the experiment and, therefore, on the general conclusion on the test item mutagenicity. Material and methods. A plate incorporation test version was used both in the presence and absence of a metabolic activation system. Results. We summarized the historical control data obtained in the laboratory in 2016-2020, determine the fluctuation limits in the number of revertant colonies for each strain, and identify the factors affecting the negative control variability. No significant differences were found in the spontaneous background of test strains when using DMSO or water as solvents, polypropylene or polystyrene tubes, as well as Petri dishes of different types. In the case of the TA1535, TA102 and TA100 cultures, no influence of the presence of the S9 mixture on the spontaneous reversion range was revealed (p≤0.05). Statistically significant differences in the number of spontaneous revertants (at + S9 or -S9) were found for the strains that allow detecting frameshift mutations, TA97 and TA98. It has been shown that the volume of the selective medium and the brand of gelling agent in its composition are important factors leading to the variability of the historical negative control. Conclusion. To ensure the quality of experiments according to the principles of good laboratory practice and the reliability of the data obtained using the bacterial reverse mutation method, it is necessary to standardize the operations in advance of experiments.

Comparison of the sensitivities of Salmonella typhimurium strains TA102 and TA2638A to 16 mutagens.

The qualitative and quantitative sensitivity of the genetically related, histidine-auxtrophic Salmonella typhimurium strains TA102 and TA2638a to 16 compounds was examined. The compounds were mainly cross-linking and oxidising mutagens, the effects of which were known to be detected by strain TA102 preferentially or by a combination of Escherichia coli WP2 (pkM101) and uvrA/pkM101. The morphology and number of spontaneous revertants was also compared. Fourteen of the 16 compounds caused reversion in both strains. Bleomycin and streptomycin induced reversion in strain TA102 but not TA2638a. The greater sensitivity of TA102 to these compounds may be associated with the extrachromosomal location of the target genes. The overall quantitative sensitivity of the two strains was similar for the other compounds. The number of compounds that caused reversions at lower doses or produced greater proportional increases were the same in TA102 as in TA2638a. The spontaneous number of revertants, without and with metabolic activation, respectively, was 98 and 130 for TA2638a and 322 and 465 for TA102. Strain TA2638a formed larger, more uniform colonies than TA102. The present results together with those of previous studies indicate a high degree of concordance between the sensitivity of strains TA102 and TA2638 for the detection of mutagens. The uniform colony size and lower spontaneous reversion frequency seen with strain TA2638a compared with TA102 would make it more reliable and convenient for routine testing. It is concluded that strain TA2638a should be considered as an alternative to TA102 and included, as well as the two E.coli strains, in the set of bacterial strains used in the standard test battery for mutagenicity testing.

Urinary mutagenicity on TA98 and YG1024 Salmonella typhimurium strains after a hamburger meal: influence of GSTM1 and NAT2 genotypes.

Mutagenicity on TA98 and YG1024 Salmonella typhimurium strains of pan-fried hamburger extracts and of 24 h post-meal urine from 32 non-smoking volunteers was evaluated. Each participant in the study was GSTM1 and NAT2 genotyped. After cooking the meat showed mutagenic activity (mean +/- SD) on strains TA98 and YG1024 of 114 +/- 129 and 1437 +/- 1536 net revertants/g respectively. Twenty three of 32 urine samples showed clear mutagenic activity (i.e. caused at least a doubling of the number of spontaneous revertants) on the O-acetyltransferase over-producing strain YG1024, while none of the post-meal 24 h urine samples was clearly mutagenic on strain TA98. Total 24 h post-meal YG1024-active urinary mutagens were well correlated with the levels of mutagen intake with the meal (r2 = 0.5977, F = 44.58, P < 0.01). In the group under study GSTM1 genotypes did not influence urinary mutagenicity. Highly exposed subjects (n = 15) with the NAT2-ss genotype showed significantly increased levels of urinary mutagenicity on strain YG1024 in comparison with NAT2-R subjects (mutagen intake-adjusted total 24 h mutagen excretion = 1.00 +/- 0.29 versus 0.66 +/- 0.32, Mann-Whitney U test, U = 12.5, P < 0.05). Our results suggest that the levels of urinary mutagens derived from diets rich in heterocyclic aromatic amines, which are specifically detected by the YG1024 Salmonella strain, are modulated by NAT2-dependent enzyme activity, slow acetylators having higher levels of mutagens in their urine. Subjects with the rapid acetylator genotype, who are known to be at risk for colon cancer, seem to be partially protected with respect to the risk of bladder cancer.

Effects of amount and type of agar on the number of spontaneous revertants in the Ames test

It has been shown that the spontaneous revertants in the Ames test can depend on the amount and type of bottom agar on the plates. The most clear-cut effect was found with the his G46 strains, carrying the ΔuvrB mutation, and with the new hisG428 strain, TA102. Evidence of mutagenic impurities in agar has been found.

A simple modification of the Salmonella liquid-incubation assay Increased sensitivity for detecting mutagens in human urine

A simple modification of the Salmonella/microsome liquid-incubation procedure improves the sensitivity of the assay for detecting mutagens in human urine. Extracts from cigarette smokers' urine were used as a model complex mutagenic mixture for validation of the assay. The modification consists of adding increased numbers of bacterial cells (approximately 10 9) in a concentrated suspension to liver homogenate mix and urine extract, all in 0.2-ml volume. After 90 min incubation at 37°C, the mixture is processed according to the standard Ames test protocol. This procedure is 20 times more sensitive than the standard plate-incorporation test and 13 times more sensitive than a previously reported liquid-incubation protocol. The number of spontaneous revertants did not increase under these conditions and, compared to the plate-incorporation test, 10-fold less liver homogenate and 5-fold less enzymatic cofactors were needed per plate. The procedure was approximately 14 times more sensitive in detecting the mutagenic activity of benzo[ a]pyrene. We also used the modification to determine mutagenic activity in urine from a group of non-smokers. The method may be generally useful for investigations of mutagenic activity in human urine samples.

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.