Analysis of chemical structures and mutations detected by Salmonella TA98 and TA100

Abstract

As part of an analysis performed under the auspices of the International Workshop on Genotoxicity Testing (IWGT) in 2017, we and others showed that Salmonella frameshift strain TA98 and base-substitution strain TA100 together + /- S9 detected 93% of the mutagens detected by all the bacterial strains recommended by OECD TG471 (Williams et al., Mutation Res. 848:503081, 2019). We have extended this analysis by identifying the numbers and chemical classes of chemicals detected by these two strains either alone or in combination, including the role of S9. Using the Leadscope 2021 SAR Genetox database containing > 21,900 compounds, our dataset containing 7170 compounds tested in both TA98 and TA100. Together, TA98 and TA100 detected 94% (3733/3981) of the mutagens detected using all the TG471-recommended bacterial strains; 39% were mutagenic in one or both strains. TA100 detected 77% of all of these mutagens and TA98 70%. Considering the overlap of detection by both strains, 12% of these mutagens were detected only by TA98 and 19% only by TA100. In the absence of S9, sensitivity dropped by 31% for TA98 and 29% for TA100. Overall, 32% of the mutagens required S9 for detection by either strain; 9% were detected only without S9. Using the 2021 Leadscope Genetox Expert Alerts, TA100 detected 18 mutagenic alerting chemical classes with better sensitivity than TA98, whereas TA98 detected 10 classes better than TA100. TA100 detected more chemical classes than did TA98, especially hydrazines, azides, various di- and tri-halides, various nitrosamines, epoxides, aziridines, difurans, and half-mustards; TA98 especially detected polycyclic primary amines, various aromatic amines, polycyclic aromatic hydrocarbons, triazines, and dibenzo-furans. Model compounds with these structures induce primarily G to T mutations in TA100 and/or a hotspot GC deletion in TA98. Both TA98 and TA100 + /- S9 are needed for adequate mutagenicity screening with the Salmonella (Ames) assay.

Keywords: Ames assay; Bacterial mutagenicity; Mutagenicity; Mutagenicity screening; Mutation spectra; Salmonella mutagenicity assay.

Figure 1.

Data flow for investigation of strain-specific data sets.

Figure 2.

Mutagens detected in TG471 strains. The set of tested mutagens detected in TG471 strains was 3,981, and the percentage detected by TA98 and/or TA100 was 94%. In this set, 2,335 mutagens were detected by TA98 and 2,954 by TA100; 1,565 mutagens were positive in both strains.

Figure 3.

Mutagens detected in TA98 and TA100. The set of mutagens tested both in TA98 and TA100 is shown, as well as the percentage detected by TA98 and/or TA100 (89%). In this set, 1,901 mutagens were detected by TA98 and 2,092 by TA100; 1,565 mutagens were positive in both strains.

Figure 4.

(A) Number and percentage of mutagens detected by TA98 +S9; 31% of the 2047 mutagens required S9 for detection. (B) Number and percentage of mutagens detected by TA100 +S9; 29% of the 2474 mutagens required S9 for detection. (C) Number and percentage of mutagens detected by TA98 and/or TA100 +S9; 32% of the 3130 mutagens required S9 for detection.

Figure 5.

(A) Number and percentage of mutagens detected by TA98 −S9; 7% of the 1879 mutagens did not require S9 for detection. (B) Number and percentage of mutagens detected by TA100 −S9; 7% of the 2032 mutagens did not require S9 for detection. (C) Number and percentage of mutagens detected by TA98 or TA100 −S9; 9% of the 2720 mutagens did not require S9 for detection.

Figure 6.

Structural Alerts of agents detected preferentially by TA98 and/or TA100; data described in Table 1. Note that when multiple representations of an alert exist, only one example is displayed.

Figure 7.

Target for reversion of the hisG46 allele in TA100 [16]. The hisG46 codon 69 is underlined; all mutants recovered involve base changes in at least the first or second C of the CCC codon. This target permits recovery of GC to TA, GC to AT, and GC to CG base substitutions. In addition, TA to GC base substitutions are also recovered at this target by mutation in either of two extragenic suppressor threonine t-RNAs: thrV[SuGGG] and thrT[SuGGG], such that the gene products of the mutant alleles, altered tRNA^Thr1 and tRNA^Thr3, suppress the his46 mutation [18]. In addition, multiple mutations can be recovered at the hisG46 allele that consist of base substitutions at the first and/or second C of the CCC codon plus additional base substitutions within the target sequence.

Figure 8.

Target for reversion of the hisD3052 allele in TA98 [17,28]; reprinted with permission. The target is a minimum of 76 base pairs long. Regions are noted within which deletions and duplications have been recovered; the largest deletion (the 35-bp filled bar) and duplication (the 46-bp open bar) recovered are shown. The hotspot region is in bold and consists of a CGCGCGCG sequence at which a 2-base deletion is recovered frequently. Complex mutations consist of a frameshift mutation (typically, the 2-base CG hotspot deletion, and an associated base substation, typically, adjacent to the hotspot CG deletion. All mutants recovered involve deletions or duplications of all or parts of the hotspot region (bold).