Mutagenicity spectra in Salmonella typhimurium strains of glutathione, L-cysteine and active oxygen species

Glutathione and L-cysteine, in the presence of rat kidney post-mitochondrialsupernatant (S9) fraction, and various forms of active oxygenwere investigated for mutagenicity in seven his^– strainsof Salmonella typhimurium. Glutathione and L-cysteine showedqualitatively and quantitatively virtually identical mutagenicactivities. The number of mutants induced in strain TA97 was3–4 times higher than in TA100, the strain in which themutagenicity was originally detected. Mutagenic effects werealso observed in strains TA92, TA102 and TA104, but not in TA1535and TA1537. Hydrogen peroxide, superoxide and glucose/glucoseoxidase in the presence and absence of kidney S9 fraction showedpronounced mutagenic effects in strains TA104 and TA102. Additionally,weak mutagenic effects were observed in TA100, while the remainingstrains, including TA97, were not responsive. These mutagenicityspectra suggest that the mutagenic species formed from glutathioneand L-cysteine are similar, if not identical, and are differentfrom hydrogen peroxide, superoxide and other oxygen speciesderived from them. Further support for this notion was givenwhen it was observed that catalase did not affect the mutagenicityof glutathione and that superoxide dismutase showed a significanteffect only when used in milligram quantities. This study showsthat mutagenicity spectra may be useful in the elucidation ofactivation pathways. Furthermore, it is interesting to notethat all the compounds and preparations showing a positive responsein the Ames test in the present study occur endogenously inorganisms: glutathione, L-cysteine, hydrogen peroxide, superoxide,glucose, glucose oxidase and kidney S9 fraction (which was mutagenicin several strains).     

The SIMULTEST: a new approach to screening chemicals with the Salmonella reversion assay

A new Salmonella mutagenicity test method is under development to test a chemical with more than one strain simultaneously (the "SIMULTEST"), that is, different Salmonella typhimurium tester strains are used in combination on the same plate. Strains are combined in two sets: strains with plasmid pKM101 (TA97, TA98, TA100, and TA102) and strains without the plasmid (TA1535, TA1537, and TA1538). The SIMULTEST combinations successfully detect the mutagenic activity of five mutagens in different chemical classes. This approach may be useful in reducing the workload associated with mutagenicity testing with Salmonella.     

Propyldazine is mutagenic in Salmonella typhimurium and Escherichia coli: distinct specificity for strains TA1537 and TA97

The antihypertensive drug propyldazine (Atensil) was demonstrated to be mutagenic with auxotrophic mutants of Salmonella typhimurium and Escherichia coli. Addition of liver S9 mix (postmitochondrial supernatant fraction supplemented with an NADPH-generating system) had little, if any, effect on the mutagenicity. The mutagenicity showed an unusual pattern of strain specificity. Increased frequencies of reversion were observed with all strains whose auxotrophy was caused by frame-shift mutations: the number of revertant colonies per plate from S. typhimurium TA98, TA1538, TA97, and TA1537 was increased up to 5-, 9-, 43-, and 160-fold, respectively, above background. Among the strains that became auxotrophic by substitution mutations, S typhimurium TA102, E. coli WP2, and E coli WP2 uvrA yielded positive results (twofold above background). S. typhimurium TA1535 and TA100 were not reverted by propyldazine. It should be noted that propyldazine, due to its low toxicity and good solubility, could be tested up to very high doses. Hence, although quite impressive mutagenic effects occurred, the mutagenic potency was moderate even in the most responsive strains, TA1537 and TA97 (about 0.3 and 1.0 revertants per nmole, respectively). With the limitation that the strain specificities were different, the mutagenic potency of propyldazine was in the same order of magnitude as that of hydralazine and dihydralazine, two related antihypertensive drugs which were already known to be mutagenic. In our hands, both compounds were mutagenic in S typhimurium TA1535, TA100, TA1537, and TA98. These results differ from data in the literature in that we found clear but weak effects even with strains for which others have reported negative results.     

The comparative responses of Salmonella typhimurium TA1537 and TA97a to a range of reference mutagens and novel compounds

Salmonella typhimurium TA97a was added to the set of indicator strains routinely used for the Ames test in this laboratory (TA1535, TA1537, TA1538, TA98 and TA100) for a trial period during which a total of approximately 40 reference mutagens and novel pharmaceutical compounds were examined. The conclusions from this trial were as follows: (i) there are agents mutagenic for TA1537 which are not detected by TA97a. (ii) except for agents requiring the R factor plasmid, TA97a shows no increased sensitivity to mutagens when compared with TA1537 and (iii) nearly all the limited published database is for the original isolate, TA97, rather than TA97a, and the results obtained here indicate significant differences in response between the two; TA97a remains, therefore, essentially unvalidated.     

Photomutagenicity assays in bacteria: factors affecting assay design and assessment of photomutagenic potential of para-aminobenzoic acid

Photomutagenicity assays are required for regulatory submissionsof some chemicals. As yet there are no wellvalidated protocolsavailable for these assays. Critical factors which may contributeto the ability of a bacterial assay to detect photomutagens(e. g. dose of UV and test chemical, exposure conditions, lightsource, bacterial strains) were investigated using two knownphotomutagens, chlorpromazine and 8-methoxypsoralen. Salmonellatyphimurium strains TA98, TA102 and TA1537 and Escherichia colistrains WP2 and WP2(pKM101) were used and differences in theresponsiveness of these strains were observed with these substances.Both chemicals were detected using either UV exposure in suspensionor on the agar plates. On the basis of these observations andon other results reported in the literature, recommendationsare made on protocol aspects for assessing photomutagenic potentialin routine screening tests. Using these recommendations thesunscreen para-amino-benzoic acid was tested in S. typhimuriumstrains TA98, TA100, TA1535, TA1537 and E. coli strains WP2and WP2(pKM101), using both plate irradiation and suspensionexposure conditions. No evidence of mutagenic potential wasdetected.     

Bacterial mutagenicity assays: Vehicle and positive control results from the standard Ames assay,the 6‐ and 24‐well miniaturized plate incorporation assays and the Ames II™ assay

Bacterial mutation assays are conducted routinely as part of the safety assessment of new chemicals. The OECD Test Guideline (TG) 471 describes the conduct of the standard agar plate Ames assay, required for regulatory submissions. Higher throughput non‐OECD 471 TG assays, such as the miniaturized plate incorporation and Ames II? assays, can be used for prescreening purposes. We have compiled historical vehicle and positive control data generated using these methods. The historical database is comprised from experiments spanning 9 years and includes >1000 experiments from the standard Ames assay using the plate incorporation and pre‐incubation methods (TA98, TA100, TA1535, TA1537, and WP2 uvrA), >50 experiments from the 6‐well (TA98, TA100, TA1535, TA97a, and WP2 uvrA) and >100 experiments from the 24‐well (TA98, TA100, TA102, TA1535, TA1537, and TA97a) plate incorporation assays, and >1000 experiments from the Ames II? assay (TA98 and TAMix). Although miniaturization to a 24‐well format made the measurement of control revertant colonies in TA1537 and TA1535 more difficult; this can be overcome by using an alternative strain with a higher spontaneous reversion rate (i.e., using TA97a instead of TA1537) or by increasing the number of replicate wells to 12 (for TA1535). All three miniaturized methods, including the Ames II? assay, were responsive to known mutagens and the responses were reproducible over years of use. These data demonstrate the excellent reproducibility of the standard and miniaturized bacterial mutation assays using positive control chemicals. Environ. Mol. Mutagen. 57:483–496, 2016. © 2016 Wiley Periodicals, Inc.     

A promising Ames battery for mutagenicity characterization of new dyes

When testing new products, potential new products, or their impurities for genotoxicity in the Ames test, the quantity available for testing can be a limiting factor. This is the case for a dye repository of around 98,000 substances the Max Weaver Dye Library (MWDL). Mutagenicity data on dyes in the literature, although vast, in several cases is not reliable, compromising the performance of the in silico models. In this report, we propose a strategy for the generation of high‐quality mutagenicity data for dyes using a minimum amount of sample. We evaluated 15 dyes from different chemical classes selected from 150 representative dyes of the MWDL. The purity and molecular confirmation of each dye were determined, and the microplate agar protocol (MPA) was used. Dyes were tested at the limit of solubility in single and concentration‐response experiments using seven strains without and with metabolic activation except for anthraquinone dyes which were tested with eight strains. Six dyes were mutagenic. The most sensitive was YG1041, followed by TA97a > TA98 > TA100 = TA1538 > TA102. YG7108 as well as TA1537 did not detect any mutagenic response. We concluded that the MPA was successful in identifying the mutagenicity of dyes using less than 12.5 mg of sample. We propose that dyes should be tested in a tiered approach using YG1041 followed by TA97a, TA98, and TA100 in concentration‐response experiments. This work provides additional information on the dye mutagenicity database available in the literature.     

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.     

Mutagenic activity in synthetic pyrethroids in Salmonella typhimurium

Four pyrethroids, allethrin, resmethrin, permethrin and fen-valerate,were tested for mutagenicity in bacterial reversion assay systemswith seven strains (TA1535, TA100, TA1538, TA98, TA1537, TA97and TA104) of Salmonella typhimurium. Our results show thatthree pyrethroids, namely resmethrin, permethrin and fenvalerate,were not found to be mutagenic in S. typhimurium in the presenceor absence of a rat liver activation system. Allethrin was foundto be mutagenic with TA100, TA104 and TA97 strains and requiredmetabolic activation (S9 mix) in order to show its activity,mainly with TA100 and TA104 strains.     

Mutagenicity,antioxidant potential,and antimutagenic activity against hydrogen peroxide of cashew (Anacardium occidentale) apple juice and cajuina

Fresh and processed cashew (Anacardium occidentale) apple juice (CAJ) are among the most popular drinks in Brazil. Besides their nutritional benefits, these juices have antibacterial and antitumor potential. The chemical constituents of both the fresh juice and the processed juice (cajuina) were analyzed and characterized as complex mixtures containing high concentrations of vitamin C, various carotenoids, phenolic compounds, and metals. In the present study, these beverages exhibited direct and rat liver S9-mediated mutagenicity in the Salmonella/microsome assay with strains TA97a, TA98, and TA100, which detect frameshifts and base pair substitution. No mutagenicity was observed with strain TA102, which detects oxidative and alkylating mutagens and active forms of oxygen. Both CAJ and cajuina showed antioxidant activity as determined by a total radical-trapping potential assay. To test whether this antioxidant potential might result in antimutagenesis, we used a variation of the Salmonella/microsome assay that included pre-, co-, and posttreatment of hydrogen peroxide-exposed Salmonella typhimurium strain TA102 with the juices. CAJ and cajuina protected strain TA102 against mutation by oxidative damage in co- and posttreatments. The antimutagenic effects during cotreatment with hydrogen peroxide may be due to scavenging free radicals and complexing extracellular mutagenic compounds. The protective effects in posttreatment may be due to stimulation of repair and/or reversion of DNA damage. The results indicate that CAJ and cajuina have mutagenic, radical-trapping, antimutagenic, and comutagenic activity and that these properties can be related to the chemical constituents of the juices.     

Microbial mutagenicity of isomeric two-, three-, and four-ring amino polycyclic aromatic hydrocarbons

The isomers of various two-, three-, and four-ring amino polycyclic aromatic hydrocarbons were tested for mutagenic activity using a microbial plate incorporation test with four Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537). All compounds were assayed with an S9 metabolic activating enzyme system. The two-ring compounds were tested only with TA98. All were weakly mutagenic (1-10 rev/micrograms) except 2-aminobiphenyl, which was not mutagenic under these test conditions. All except two of the 13 fused three-ring compounds (aminofluorenes, aminoanthracenes, and aminophenanthrenes) were active frame shift mutagens; only the aminophenanthrenes were active base-pair mutagens. The potency of this group of isomeric compounds ranged from moderately (approximately 20 rev/microgram) to strongly (greater than 5,000 rev/microgram) mutagenic. As a group, the pericondensed four-ring amino compounds were the most mutagenic of the three groups tested. All of the aminofluoranthene and aminopyrene isomers showed significant mutagenic activity with TA98, TA100, and TA1537. In general, the mutagenic potency of the amino polycyclic aromatic compounds tested was highly dependent on the structural position of the amino group.     

Formaldehyde is a bacterial mutagen in a range of Salmonella and Escherichia indicator strains

Formaldehyde was examined for bacterial mutagenicity using Escherichiacoli WP2(pKM101) and WP2uvrA(pKM101), and Salmonella typhimuriumTA1535, TA1537, TA1538, TA98, TA100 and TA102, in the absenceof any exogenous source of metabolic activation. Using pre-incubationexposure, clear mutagenicity was seen for TA98, TA100 and TA102,and both E.coli strains. In standard plate-incorporation assays,consistent mutagenicity was seen only for TA100 and WP2uvrA(pKM101).No evidence of mutagenicity was seen for TA1535, TA1537 or TA1538using either method of exposure. These data confirm the enhancedability of the pre-incubation method to detect the mutagenicityof formaldehyde both quantitatively, as expressed by numbersof revertant colonies, and qualitatively, in terms of the rangeof indicator strains reverted. The relatively greater sensitivityof the pre-incubation assay is probably due to better containmentof a volatile agent and/or lack of interaction with agar duringthe initial period of exposure. The findings are consistentwith the suggestion that formaldehyde induces lesions in bacteriawhich are, at least to some extent, excision-repairable, andindicate that the presence of the R-factor plasmid may be requiredfor the expression of its mutagenicity in excision repair-deficientSalmonella.     

Genotoxic activity in microorganisms of tetryl, 1,3-dinitrobenzene and 1,3,5-trinitrobenzene

N-Methyl-N,2,4,6-tetranitroaniline (tetryl), 1,3-dinitrobenzene, and 1,3,5-trinitrobenzene were subjected to DNA repair assays using the Escherichia coli W3110/polA⁺, p3478/polA^? system, reverse mutation assays with His^? Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, and mitotic recombinogenic tests with the yeast Saccharomyces cerevisiae D5. Tests were carried out in the absence of an exogenous activation system and in tissue homogenate-mediated assays using Aroclor 1254-induced, male rat-liver-derived S9 mix. Mutagenic activity of tetryl was demonstrated with S typhimurium strains TA1537, TA1538, TA98, and TA100. The responses were particularly strong in the absence of S9 mix. Tetryl also induced increases in recombinant numbers and frequencies in the S cerevisiae test without the S9 mix, but not in its presence. 1,3-Dinitrobenzene was demonstrated to be a mutagen with S typhimurium strains TA1538, TA98, and TA100. Slight activity was also seen with TA1537. The S9 mix reduced the magnitude of the responses. 1,3,5-Trinitrobenzene was also demonstrated to be mutagenic with S typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100. Again, the S9 mix reduced the magnitude of the responses. In this segment of a programme initiated by military authorities, the genotoxic potential of three nitroaromatic compounds, which have found significant use in explosive preparations, has been demonstrated. Twelve other compounds used in ordnance were not active in any of the test systems. These were octahydro-1-acetyl-3,5,7-trinitro-S-tetramine (SEX), hexahydro-1,3-dinitro-5-acetyl-S-triamine (TAX), ethyl centralite, 2-nitrodiphenylamine, N-nitrosodiphenylamine, diphenylamine, diethyleneglycoldinitrate, nitroguanidine, lead salicylate, lead resorcylate, red phosphorus, and zinc chloride.     

四种医用高分子材料的Ames试验研究

本文报道用Ａｍｅｓ法检测４种医用高分子材料的致突变性。结果表明，经ＴＡ９７，ＴＡ９８，ＴＡ１００和ＴＡ１０２菌株测试的复合膜，胶原膜，铬复合缝线和复合缝线各剂量在点试验和平皿掺入试验中未诱发阳性反应。但是４个菌株所对应的突变剂均诱发了阳性突变。因此４种医用高分子材料在本实验条件下无诱发突变作用。     

Mutagenic screening of marker grenade dyes by the Salmonella reversion assay, L5178Y/TK+/- mouse lymphoma assay, and in vivo sister chromatid exchange analysis in mice

Two dyes (C.I. Solvent Yellow No. 33 and a mixture of C.I. Solvent Yellow No. 33 and C.I. Solvent Green No. 3) were tested for mutagenicity in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay, and also for sister chromatid exchange (SCE) induction in vivo in C57B1/6J mice. In addition, a greater than 99.9% pure sample of the yellow dye [2-(2'-quinolyl)-1,3-indandione] was tested with and without exogenous activation in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay. Neither C.I. Solvent Yellow No. 33 nor the C.I. Solvent Yellow No. 33 and Solvent Green No. 3 mixture was positive for inducing SCEs in vivo. All three dyes were tested in the standard plate incorporation test in seven Salmonella strains TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. The dyes were negative with and without exogenous activation in TA98, TA1535, and TA1538. One test with TA1537 was positive with the greater than 99.9% purified yellow dye. All three dyes gave weakly positive results (less than a twofold increase) with S-9 in TA100 and were clearly positive in TA102 and TA104 both with and without S-9. They also induced mutation at the thymidine kinase locus in mouse lymphoma cells, produced both large- and small-colony trifluorothymidine-resistant mutants, and were clastogenic. The purified yellow dye was further tested for SCE induction in mouse lymphoma cells and was determined to give a slightly positive response in the presence of S-9.     

SOS induction in Escherichia coli and Salmonella mutagenicity: a comparison using 330 compounds

To examine the concordance of two microbial genotoxicity short-termassays, 330 experimental results for the SOS chromotest usingtester strain Escherichia coli PQ37 were compared with the resultsof the Salmonella/mammalian microsome mutagenicity assay withSalmonella typhimurium TA97, TA98, TA100, TA102, TA104, TA1535,TA1537 and/or TA1538. With respect to qualitative features,the concordance between SOS chromotest and Salmonella mutagenicitytest results was 86.4% (sensitivity, 78.6%; specificity, 100%;     

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。     

Comparison of Salmonella typhimurium TA102 with Escherichia coli WP2 tester strains

In 1982, Levin et al. published a paper describing a new Salmonella typhimurium strain, TA102, for detecting mutagenic agents that react preferentially with AT base pairs. This strain has an AT base pair at the critical mutation site within the hisG gene, which is located on a multicopy plasmid, pAQ1; the chromosomal copy of the hisG gene has been deleted. It also has an intact excision repair system, thus facilitating the detection of cross-linking agents, and carries the mutator plasmid, pKM101. Although TA102 has been shown to be reverted by certain mutagenic agents that are not detected in the usual battery of strains (TA1535, TA1537, TA1538, TA98 and TA100), there has been a general reluctance within the field to include TA102 as one of the standard screening strains. This may in part result from the difficulties which have been experienced in many laboratories in maintaining the strain, and in obtaining reproducible spontaneous and induced revertant counts. At Glaxo we routinely include certain Escherichia coli strains in our microbial test battery, and were aware that some of the genetic features offered by TA102 were already being covered by these strains. For example, E.coli WP2 (pKM101) has an AT base pair at the critical mutation site within the trpE gene, is excision proficient (and thus will detect cross-linking agents) and carries the pKM101 plasmid to enhance error-prone repair. From the published literature it was apparent that a number of the 'TA102 specific' mutagens could be detected in E.coli e.g. neocarzinostatin, UV and 8-MOP plus UV.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 SIMULTEST approach for testing mutagens in the Salmonella microtitre fluctuation assay

The concept of combining several histidine-dependent Salmonella strains in a single test, the SIMULTEST, has been applied to the microtitre fluctuation test. The activity of five mutagens was determined in strains TA97, TA98, TA100, and TA102 individually as well as in a SIMULTEST mixture. All five compounds were mutagenic in the SIMULTEST, demonstrating the utility of this time and labour-saving approach of combining strains for testing with this method. The microtitre fluctuation SIMULTEST results were quantitatively comparable to those of the SIMULTEST Salmonella/microsome plate test. The microtitre fluctuation test compared with the plate incorporation assay generally showed more favourable "sensitivity" and "quantity" indices in that four of the five chemicals tested in the fluctuation test were mutagenic at lower doses than in the plate test.