Curing and induction of the Fels 1 and Fels 2 prophages in the Ames mutagen tester strains of Salmonella typhimurium

A method is described for curing the Ames Salmonella mutagen tester strains of their Fels 1 and Fels 2 prophages with the aid of the antitumor drug daunorubicin. Non-lysogenic derivatives corresponding to TA100 and TA1535 were isolated and designated TAQ100 and TAQ1535 respectively. In addition, the Fels 1 monolysogens TAQ100F1 and TAQ1535F1, as well as the Fels 2 monolysogens TAQ100F2 and TAQ1535F2, were obtained. Finally, strains corresponding to TA98 and TA1538 cured of Fels 2, but retaining a cryptic Fels 1 (F1d) prophage were isolated and designated TAQ98F1d and TAQ1538F1d respectively. The various cured derivatives were identified by colony hybridization with ³²P-labeled probes of Fels 1 and Fels 2 DNA. Southern blot hybridizations confirmed that phage-specific Fels DNA sequences were missing from the cured strains. The Fels 2-cured strains were resistant to Fels 2, but Fels 1 grew, albeit poorly, on the Fels 1-cured strains. Strains TAQ100F1, TAQ1535F1, TAQ100F2 and TAQ1535F2 were used in prophage induction assays, in the presence of rat-liver extract where necessary. Daunorubicin, bleomycin, mitomycin C, aflatoxin B1, 2-amino-dipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-2) were found to induce Fels 1 and/or Fels 2 in at least one of these strains. The induction of the Fels prophages in the TAQ monolysogens may provide a useful complement to the Ames test for the detection of DNA-damaging agents and potential carcinogens.     

A comparative study of mutagenic and SOS-inducing activity of biphenyls, phenanthrenequinones and fluorenones

A total of 23 chemicals--biphenyls, phenanthrenequinones and fluorenones--were tested for mutagenicity towards Salmonella typhimurium strains TA1538, TA1535 and TA98. SOS-inducing activity of the same chemicals was studied in terms of the SOS-inducing potency in Escherichia coli PQ37, using an automated instrument controlled by a dedicated computer program for the SOS Chromotest. Of the 23 chemicals studied 14 induced His+ revertants in S. typhimurium TA1538 hisD305 (-1 frameshift); none induced His+ reversions in TA1535 (base-pair substitution). The mutagenicity of the chemicals in S. typhimurium TA98 (pKM 101) was lower than in TA1538. There was a close correlation between mutagenicity and SOS-inducing activity of fluorenones and phenanthrenequinones. None of the biphenyls tested induced SOS response and this property does not depend upon the mutagenic activity of the chemicals. SOS Chromotest is particularly valid in detecting chemicals which give rise to base-pair substitutions through SOS induction. If positive results are obtained, the Salmonella assay may be omitted. However, this test cannot replace the Ames test especially for the primary screening of mutagenicity of chemicals with unknown structure.     

Radiation-induced mutagenicity and lethality in ames tester strains of salmonella

Mutation and killing induced by X radiation and 60CO gamma radiation were studied in six different histidine-requiring auxotrophs of Salmonella typhimurium. Strain TA100, which is sensitive to base-pair substitutions, and strains TA2637 and TA98, which are sensitive to frameshifts, carry the pKM101 plasmid and exhibit significantly higher radiation-induced mutations compared to their plasmidless parent strains TA1535, TA1537, and TA1538, respectively. Among the plasmid-containing strains, TA98 and TA2637 are much more sensitive to the mutagenic action of radiation than is TA100 based on a comparison with their respective spontaneous mutation rates; however, no uniformity was observed in the responses of the strains to the lethal action of ionizing radiation. The pKM101 plasmid provides partial protection against lethality in TA100 and TA2637, whereas the same plasmid enhances the lethal action of ionizing radiation in TA98. The following conclusions are consistent with these observations: (1) the standard Ames Salmonella assay correctly identifies ionizing radiation as a mutagenic agent; (2) frameshift-sensitive parent strains are more sensitive to the mutagenic effects of ionizing radiation than is the only strain studied that is sensitive to base-pair substitutions; and (3) enhancement of mutagenesis and survival is related to plasmid-mediated repair of DNA damage induced by ionizing radiation and does not involve damage induced by Cerenkov-generated uv radiation which is negligible for our irradiation conditions.     

Comparative mutability of the Ames tester strains of Salmonella typhimurium by ultraviolet radiation and by 4-nitroquinoline 1-oxide

A standard method for determining mutant frequencies per survivor was used to study the detailed kinetics of reverse mutations of Ames tester strains of Salmonella typhimurium induced by UV and by 4N1O. After UV irradiation, strain TA1538 was non-mutable, but its plasmid-containing derivative TA98 was mutable, whereas TA1535 was mutable and its plasmid-bearing derivative TA100 was about 10-fold more mutable. After treatment with 4NQO, TA98 was less mutable than TA1538, whereas TA100 was more mutable than TA1535 by a factor of 10–50. TA1537 was slightly less mutable than TA1535 by either UV or 4NQO. The differential mutabilities of these strains are briefly discussed in relation to the “hot spot” base sequences for reversion and the nature of DNA damage caused by UV and 4NQO.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

Measurement of drug-metabolizing systems in Salmonella typhimurium strains G46, TA1535, TA100, TA1538 and TA98

Salmonella typhimurium strains which are commonly used in the Ames test for screening potential carcinogens were examined for a number of drug-metabolizing systems. Neither cytochrome P-450 itself nor two activities catalyzed by the cytochrome P-450 system in mammalian cells, i.e., benzpyrene monooxygenase and ethoxycoumarin O-deethylation, could be detected. Nor do these bacterial strains demonstrate any ability to detoxify epoxides by hydrating them or to conjugate p-nitrophenol with glucuronic acid.On the other hand, S. typhimurium strains G46, TA1535, TA100, TA1538 and TA98 contain considerable amounts of acid-soluble thiols, approx. 5–10% of which is glutathione. These bacteria can also enzymatically conjugate glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) and can reduce oxidized glutathione using NADPH as cofactor.Thus, enzymatic and non-enzymatic reaction of immediate carcinogens with thiol groups in S. typhimurium may have a significant effect on the outcome of the Ames test in certain cases.     

Isolation and characterization of an isogenic set of Salmonella typhimurium strains analogous to the "Ames" tester strains

The standard Ames tester strains of Salmonella typhimurium contain a number of genetic differences at loci other than his. The fact that these strains contain independently isolated uvrB-bio-gal deletions and rfa mutations implies that these are likely to vary from strain to strain. Since the strains were isolated from different parental stocks of S. typhimurium LT-2, they differ in their ability to metabolize arabinose. Other, unknown differences may exist because the isolation of some of the strains involved ultraviolet and chemical mutagenesis. We have isolated a set of isogenic S. typhimurium strains that contain the relevant genetic markers of the standard Ames tester strains. These strains all contain the same uvrB-bio-gal deletion and the same rfa mutation; they differ only in the nature of their his mutations and in the presence or absence of the plasmid pKM101. We have assessed the responsiveness of these strains to a number of mutagens and conclude that their mutagenic specificities are the same as those of the corresponding Ames strains: TA98, TA100, TA1535, TA1537 and TA1538. Therefore, the specificity of the standard Ames strains with respect to these mutagens is a result solely of the differences in the nature of their his mutations and the effects of pKM101.     

Revised methods for the Salmonella mutagenicity test

The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed.     

Mutagenicity testing with TA97 and TA102 of 30 DNA-damaging compounds, negative with other Salmonella strains

In a comparative study on 135 compounds of various chemical classes, 30 agents inducing direct nonreparable DNA damage in repair-deficient E. coli failed in reverting strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium (De Flora et al., 1984b). These compounds were re-assayed in the Ames test using strains TA97 and TA102. A dose-dependent mutagenic response was detected with aminoantipyrine and p-rosaniline in TA97 and with streptomycin and formaldehyde in TA102. p-Rosaniline was the only mutagen requiring metabolic activation. 5 compounds, i.e. o-aminophenol in TA97 and methanol, ethanol, cadmium chloride and cadmium sulfate in TA102, induced a reproducible increase in revertants over controls, but this was less than 2-fold. The remaining 21 chemicals--including amino compounds, aliphatics, aromatics, heterocycles, hydrazine derivatives and inorganics--confirmed their inactivity in the Ames test. Overall data for 135 compounds, comparing the Ames test (7 strains) and the DNA-repair test (3 strains), are re-assessed on the basis of these findings.     

Mutational studies with diquat and paraquat in vitro.

Diquat and paraquat were assayed in the following tests. (1) Ames test in Salmonella typhimurium (strains TA1535, TA1537, TA1538, TA98 and TA100) with and without rat-liver microsomal fractions. (2) Resistance to 8-azaguanine in Salmonella typhimurium (strain hisG46, TA92 and TA1535. (3) Repair test in Salmonella typhimurium (strains TA1538 and TA1978). (4) Gene mutations in Aspergillus nidulans: 8-AG resistance and methionine suppression (meth A1 locus). (5) Lethal recessive damage in Aspergillus nidulans. (6) Unscheduled DNA synthesis (UDS) in human epithelial-like cells (EUE). Diquat and paraquat were positive in S. typhimurium (in the repair test and the 8-AG resistance system), in A. nidulans (for gene mutations and lethal recessive damage induction) and in EUE cells (UDS induction).     

Platinum(II) complexes generate frame-shift mutations in test strains of Salmonella typhimurium.

cis-Diamminodichloroplatinum(II) (cis-PDD) and diaquoethylenediamineplatinum(II) induce histidine revertants in Salmonella typhimurium strains TA98 (frame-shift mutation) and TA100 (base-pair substitution mutation). A linear dose--response relationship is found with cis-PDD acting on TA98 and TA100. Salmonella typhimurium strains TA1535, TA1537 and TA1538 are not sensitive to the mutagenic action of cis-PDD. All 5 strains are sensitive to the toxic effect of cis-PDD. Platinum(II) complexes induce mutations (frame-shift or base-pair substitution) only in strains carrying the R-factor plasmid.     

Comparative direct-acting mutagenicity of 1- and 2-nitropyrene: Evidence for 2-nitropyrene mutagenesis by both guanine and adenine adducts

The mutations and DNA adducts produced by the environmental pollutant 2-nitropyrene were examined in Salmonella typhimurium tester strains. 2-Nitropyrene was a stronger mutagen than its extensively studied structural isomer 1-nitropyrene in strains TA96, TA97, TA98, TA100, TA102, TA104 and TA1538. Both 1- and 2-nitropyrene were essentially inactive in TA1535. The mutagenicity of 1- and 2-nitropyrene in TA98 was much higher than in TA98NR and the activity of these compounds in TA100 was much higher than in TA100NR. While 1-nitropyrene exhibited similar mutagenicity in strains TA98 and TA98/1,8-DNP₆, the mutagenicity of 2-nitropyrene in TA98/1,8-DNP₆ was much lower than in TA98. Analysis of DNA from TA96 and TA104 incubated with 2-nitropyrene indicated the presence of two adducts, N-(deoxyguanosin-8-yl)-2-aminopyrene and N-deoxyadenosin-8-yl)-2-aminopyrene. The results suggest that 2-nitropyrene is metabolized by bacterial nitroreductase(s) to N-hydroxy-2-aminopyrene, and possibly by activation to a highly mutagenic O-acetoxy ester. DNA adduct formation with deoxyguanosine and deoxyadenosine correlates with the mutagenicity of 2-nitropyrene in tester strains possessing both G:C and A:T mutational targets.     

Comparative lethal effects of uv and ionizing radiation in Ames tester strains of Salmonella

In the three (parent-daughter) pairs of Ames Salmonella tester strains TA1535-TA100, TA1537-TA2637, and TA1538-TA98 in which the daughter strains carry the pKM101 plasmid but the parent strains do not, the pKM101 plasmid uniformly confers resistance of the host to uv radiation which indicates that the muc genes of the plasmid are present and function correctly in all three daughter strains. This uniform protection against killing by uv contrasts with the lethality responses of the same parent-daughter pairs to ionizing radiation (ir) where pKM101 again confers lethality protection to TA100 and TA2637 but sensitizes TA98 toward the lethal effects of ir. From these results we conclude that the pathways for error-prone repair of lethal lesions induced by uv and by ionizing radiation are not the same and that the muc genes of the plasmid alone are not sufficient to carry out error-prone repair of lethal lesions induced by ionizing radiation. We infer that a segment of plasmid DNA that is present in TA100 and TA2637 and is required to repair potentially lethal damage induced by ir is deleted in TA98.     

Salmonella typhimurium mutagenicity tester strains that overexpress oxygen-insensitive nitroreductases nfsA and nfsB

We have designed and constructed a series of plasmids that contain the major and/or minor Escherichia coli nitroreductase genes, nfsA and nfsB, in different combinations with R plasmid mucA/B genes and the Salmonella typhimurium OAT gene. The plasmid encoded gene products are necessary for both the metabolic activation of a range of structurally diverse nitrosubstituted compounds, and for mutagenic translation bypass. Introduction of these plasmids into S. typhimurium TA1538 and TA1535 has created several new tester strains which exhibit an extremely high mutagenic sensitivity and a broad substrate specificity towards a battery of nitrosubstituted test compounds that included 4-nitroquinoline-1-oxide (4-NQO), nitrofurazone (NF), 1-nitropyrene (1-NP), 2-nitronaphthalene (2-NN), 2-nitrofluorene (2-NF), and 1,6-dinitropyrene (1,6-DNP). Our studies show that the nfsA gene encodes a product that is extremely effective in the metabolic activation of a range of structurally diverse nitrosubstituted compounds. Several of the new tester strains are more than two orders of magnitude more sensitive to nitrosubstituted compounds than the Ames tester strains TA100 or TA98. In addition to enhancing mutagenic sensitivity, plasmids encoding both metabolic and mutagenesis functions on a single plasmid provide considerable flexibility for future mechanistic studies or tester strain development, in which it may be necessary to introduce additional plasmids containing different antibiotic resistance markers.     

Mutagenicity evaluation of HC Blue No. 1 and HC Blue No. 2. III. Effects in the Salmonella typhimurium/Escherichia coli reversion assay and the mouse lymphoma L5178Y TK+/- forward mutation assay

The hair-dye ingredients, HC Blue No. 1 (HCB1) and HC Blue No. 2 (HCB2), were tested for the induction of bacterial mutation using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100; and Escherichia coli strains WP2uvrA-. In addition, both dyes were evaluated in the mouse lymphoma L5178Y TK+/- assay (MLA) for the potential to induce forward mutation. A liver homogenate (S9) prepared from Aroclor 1254-induced male Fischer 344 rats was used to provide a means for metabolic activation. HCB1 was not mutagenic in the Ames assay, but was weakly mutagenic in the MLA only in the presence of metabolic activation. In contrast, HCB2 was a strong mutagen in the Ames assay in tester strain TA98 both in the presence and absence of metabolic activation. A positive response was also noted with HCB2 in the MLA, both in the presence and absence of metabolic activation. Negative findings from the Ames assay of this study agree with other published results where an identical lot of HCB1 was used. Using the same lot, a weak positive result was observed in the MLA, however, the activation requirements and magnitude of the response were different from that of a lot evaluated by the NTP. In contrast, HCB2 appears to be both a bacterial and mammalian cell mutagen independent of lot variability.     

Mutagenicity of the metabolites of the epoxide-diol pathway of safrole and its analogs. Study on Salmonella typhimurium]

Mutagenicity of the metabolites of the expoxide-diol pathway of safrole and analogues was studied on Ames' strains with Ames' method. Safrole, eugenol, eugenolmethylether, estragol, allylbenzene and 1'-hydroxysafrole, are not mutagen on TA 1535, TA 100 (point mutation) and TA 1537, TA 1538, TA 98 (frameshift mutations) without activation system. The corresponding epoxides that we have synthetized, are mutagens and inducers of point mutation in TA 1535 and TA 100. Dose-effect curves show differences between the mutagen efficiencies of these epoxides probably in relation with their electrophilic properties. On the other hand the 2', 3'-dihydro-2',3'-dihydroxisafrole was not mutagen in Ames' test. These results confirm the promutagen character of safrole and analogues and the role of the epoxides as proximate carcinogens.     

Glutathione and glutathione S-transferases in the salmonella mammalian-microsome mutagenicity test

Levels of the tripeptide glutathione (GSH) and the activity of glutathione S-transferases were investigated in S9 fractions of rats and mice and in Salmonella typhymurium tester strains TA1535, TA100, TA1538 and TA98. The S9 and Salmonella typhimurium tester strains had high levels of glutathione. Compared with S9, the activity of GSH S-transferases was lower in the bacteria. However, electrophiles such as 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and styrene oxide were effectively bound to bacterial GSH.

The mutagenicity of the direct mutagen CDNB was drastically lowered in presence of S9 fractions but not in presence of microsomes. A comparable decrease was obtained when microsomal supernatant, which contains GSH and GSH S-transferases, was added to the microsomes. Addition of GSH in excess completely abolished mutagenicity of CDNB. These results demonstrate that the conjugation of electrophiles with GSH mediated by the S9 fraction or the bacterial tester strains represents an important detoxication mechanism which may influence the results obtained with the Salmonella typhimurium mammalian-microsome mutagenicity test.     

The Salmonella mutagenicity test: evaluation of 29 drug candidates

The Salmonella mutagenicity test (Ames assay) is part of the routine screening battery applied to all new drugs at The Upjohn Company. The purpose of this paper is to report results for 29 compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of known biological activity and other reasons. None of the compounds reported here produced an increase in revertant colonies in the Salmonella strains employed (TA98, TA100, TA1535, TA1537 and TA1538) and therefore the Salmonella mutagenicity results with these materials do not suggest potential for mutagenesis or carcinogenesis.     

Bacterial mutagenicity of aceanthrylene: a novel cyclopenta-fused polycyclic aromatic hydrocarbon of low molecular weight

Aceanthrylene, a non-alternant cyclopenta-fused hydrocarbon, was shown to be weakly mutagenic without S9 and strongly mutagenic with S9 in the Ames Salmonella plate incorporation assay. The compound was most active in strain TA100 (35 revertants/nmole in the presence of 0.3 mg of S9 protein), and less active in strains TA98, TA1537 and TA1538 (20, 10 and 3.1 rev/nmole respectively, + S9). Strain TA1535 was unresponsive, suggesting that this compound induces frameshift mutations rather than base-pair substitutions. The mutagenic potency of aceanthrylene is consistent with predictions of its activity based on the relatively large delocalization energy (delta E deloc/beta = 0.931) of the carbonium ion which would result from oxirane ring opening of the 1,2-epoxide, a potential active metabolite.     

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs

Quercetin, rhamnetin, isorhamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation.Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3′ and 4′ positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.