Mutagenicity testing of benomyl, methyl-2-benzimidazole carbamate, streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium in vitro and in rodent host-mediated assays.

The fungicide benomyl and its commercial preparations Fundazol 50WP and Benlate 50WP and the benomyl metabolite methyl-2-benzimidazole carbamate and its commercial preparation MBC 50WP were tested for mutagenicity in in vitro spot tests, in microsomal plate assay, in liquid-culture treatments, or in rodent host-mediated assay. The base-pair substitution Salmonella typhimurium mutant hisG46 and the hisG46-bearing uvrB excision-repair-deficient mutants TA100, TA1530, TA1535 or TA1950 were used as test organisms. Complete genotypic information of these mutants is given in Ames et al. [2]. Captain 50WP, streptozotocin (SZN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-aminopurine and N-acetylaminofluorene were used as positive control compounds. In nonoverlay spot tests Benlate 50WP was not mutagenic over a dose range of 50-5000 microgram/spot in hisG46 and TA1535. In overlay spot tests 50 or 100 microgram/spot Benomyl, MBC, Fundazol 50WP, Benlate 50WP and MBC 50WP were tested in hisG46, TA1530 or TA1950. Only a non-commercial MBC sample at 100 microgram/spot showed weak mutagenic activity in hisG46. In microsomal activation plate assay MBC, benomyl, Fundazol 50WP and Benlate 50WP were tested in TA100 over a dose range of 50-2000 microgram/plate. None of the compounds showed mutagenicity. In a 20-h liquid-culture treatment 10, 100, 1000 and 10 000 microgram/ml Fundazol 50WP were not mutagenic in TA 30. In 1-h liquid-culture treatments benomyl, Benlate 50WP or Fundazol 50WP failed to induce mutations in hisG46, TA100 or TA1950 over a dose range of 0.25-1000 microgram/ml. Appropriate positive controls were mutagenic in each experiment. The consistently negative results in this study with commercial MBC and benomyl preparations are contrary to positive results reported earlier with similar methods and similar commercial preparations. Possible reasons to explain the different results are presented. The alkylating agents SZN and MNNG induced fewer mutations in TA1530 and TA1950 uvrB excision-repair-deficient strains than in the hisG46 excision-proficient strain, indicating that with these mutagens excision-repair is also a mutation-prone process. In rodent host-mediated assays with Fundazol 50WP in mice 3 consecutive subcutaneous hourly doses of 500 mg/kg in hisG46 and TA1950 and in rats or mice an oral dose of 4000 mg/kg in TA1950 were not mutagenic. The positive control SZN was mutagenic.     

Preferential induction of AT-TA transversion, but not deletions, by chlorambucil at the hisG428 site of Salmonella typhimurium TA102.

The chemotherapeutic agent chlorambucil effectively induces deletion mutations in mouse germ cells. The possibility that this chemical also effectively induces deletion mutations in bacterial DNA was examined using Ames Salmonella tester strains. Chlorambucil was mutagenic only to strains TA102 (hisG428, rfa/pKM101) and YG2975 (hisG46, rfa/pKM101) when S9 mix was absent. Since strain TA102 can detect short deletions, the mutational changes of TA102 induced by this agent without S9 mix were directly determined by the DNA sequencing technique. It turned out that chlorambucil did not induce deletion mutations but preferentially induced AT-TA transversions at the hisG428 site of plasmid pAQ1 of strain TA102. These results caution that the positive results induced by chlorambucil in mutagenicity tests do not necessarily mean the occurrence of deletions.     

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 delta uvrB mutation, and with the new hisG428 strain, TA102. Evidence of mutagenic impurities in agar has been found.     

Plasmid-dosage effects on ultraviolet, visible light and methyl methanesulfonate mutagenesis in Salmonella typhimurium

Salmonella typhimurium LT2 strains bearing plasmids pKM101, R64 or pColIb-P9 demonstrated enhanced UV survival when compared with strains not bearing plasmids. A strain of S. typhimurium bearing both pKM101 and pColIb-P9 survived UV irradiation slightly better than either of the single-plasmid strains. Spontaneous reversion of the hisG46 and trpE8 missense alleles was enhanced in each single-plasmid strain, and for the dual-plasmid strain containing pKM101 and pColIb-P9 enhancement represented a near additivity of the response seen for the single-plasmid strains. Following exposure to UV or visible-light irradiation, reversion of hisG46 and trpE8 was also enhanced in each single-plasmid strain, but quantitatively greater in the dual-plasmid strain and was equal to or slightly greater than additive the responses of the single-plasmid strains. In contrast to visible-light irradiation, UV exposure resulted in two phenotypic Trp+-revertant classes. One Trp+ class, having normal colony size (2.0 mm) and similar in number to His+ revertants, was comprised of intragenic revertants of trpE8, while the predominant Trp+ class, having smaller colony size (0.8 mm), represented intergenic suppressor revertants, illuminating the differences in mutation and/or repair specificity for UV and visible-light exposure. Methyl methane-sulfonate (MMS)-induced reversion of hisG46 was similar in effect to that seen with UV or visible-light irradiation. Plasmids pKM101 or pColIb-P9 enhanced the frequency of hisG46 reversion, while a more than additive response was seen in a strain with both plasmids. Furthermore, MMS-induced reversion of hisG46 was also observed to be greatest in a strain bearing plasmid R64 (incompatibility group I alpha) and pKM101, when compared with single-plasmid strains bearing either R64 or pKM101.     

In vivo conversion of sodium azide to a stable mutagenic metabolite in Salmonella typhimurium.

Salmonella typhimurium TA1530 and G46 strains growing in minimal medium supplemented with sodium azide produce a stable mutagenic metabolite which is not azide. The production of this metabolite is restricted to the log phase of bacteria grown in the presence of azide. The metabolite is highly mutagenic in DNA-repair defective base-substitution strains TA1530 and TA1535, but ineffective in frameshift strains TA1538 and TA1537. The metabolite induces mutations in resting cells of the TA1530 strain.     

Mutagenicity of vinyl chloride,chloroethyleneoxide, chloroacetaldehyde and chloroethanol

Exposure of S. typhimurium strains TA 1530, TA 1535 and G-46 to vinyl chloride increased the number of his⁺ rev./plate 16, 12 or 5 times over the spontaneous mutation rate. The mutagenic response for TA 1530 strain was enhanced 7, 4 or 5-fold when fortified S-9 liver fractions from humans, rats or mice were added. In TA 1530 strain, chloroacetic acid showed only toxic effects, while chloroacetaldehyde, chloroethanol and chloroethyleneoxide caused a mutagenic response. The latter compound was shown to be a strong alkylating agent.     

Methyl methanesulphonate (MMS) is clearly mutagenic in S. typhimurium strain TA1535; a comparison with strain TA100

No mutagenicity or an uncertain mutagenic response has been reported in the literature for methyl methanesulphonate (MMS) in S. typhimurium strain TA1535 when using the plate assay. In our studies we found a reproducible mutagenic activity of 62 revertants/mumole and plate for MMS in strain TA1535 when using the preincubation assay. A dose-dependent increase in revertants was, however, observed only at fairly high doses (exceeding 4 mumole). Two different slopes were observed in the dose-response curve when testing MMS with strain TA100. Slope A is dependent on the error-prone response, possible only in strain TA100 due to the pKm101 plasmid (R factor) but not possible in strain TA1535 due to its umuDC deficiency. Slope B observed at higher doses (as in strain TA1535) could be explained through a GC----AT transition initiated by the O6-methylation of guanine. Our findings demonstrate that MMS induces back mutation in S. typhimurium strains carrying the hisG46 missense mutation due to the formation of O6-methylguanine. In the case of strain TA100 the pKm101 plasmid-mediated error-prone mechanism is, however, the predominant process in MMS mutagenesis which leads to a higher mutagenic response at much lower doses than the GT----AT transition in strain TA1535.     

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).     

Localized hydroxylamine mutagenesis, and cotransduction of threonine and lysine genes, in Streptomyces venezuelae

A lysate of the generalized transducing phage SV1, grown on the prototrophic type strain 10712 of Streptomyces venezuelae, was mutagenized with hydroxylamine and used to transduce a lysineless auxotroph to lysine independence on supplemented minimal agar. A complex threonine mutant, strain VS95, was isolated from among the transductants and was shown to be carrying at least two different thr mutations. These were about 50% cotransducible with alleles of four independently isolated lysA mutations, as were two other independently isolated threonine mutations, thr-1 and hom-5. The location of thr genes close to lysA occurs in at least three other streptomycetes, but apparently not in Streptomyces coelicolor A3(2), in which the lysA and thr loci are at diametrically opposite locations on the linkage map. This first observation of cotransduction between loci governing the biosynthesis of different amino acids in the genus Streptomyces demonstrates the feasibility of fine-structure genetic analysis by transduction in these antibiotic-producing bacteria.     

Synthesis and enantioselective mutagenicity of azidoalanine in Salmonella typhimurium

Azide mutagenicity involves the requisite formation of the putative novel aminoacid metabolite, beta-azidoalanine. The role of this metabolite, however, is unclear. In order to confirm the identity of this metabolite and provide additional information on possible stereochemical requirements for mutagenicity, authentic racemic and L-azidoalanine were synthesized by an unambiguous route and tested for mutagenicity in Salmonella typhimurium TA100, TA1535, hisG46 and Escherichia coli WP2-. A marked antipodal potency ratio was observed in strains TA100 and TA1535 when racemic and L-azidoalanine were compared. The mutagenic activity resided primarily in the L-isomer. The molar potency of L-azidoalanine in TA100 and TA1535 was nearly identical to that of azide. The lack of mutagenic response for racemic or L-azidoalanine in hisG46 and E. coli WP2- was like that reported for azide and is consistent with similar modes of action for these agents.     

Chromosomal map location of the methicillin resistance determinant in Staphylococcus aureus.

Three-factor genetic crosses performed by transformation have shown that the methicillin resistance determinant of Staphylococcus aureus strain DU4916 (the mec-4916 marker) is linked to a novobiocin resistance (Novr) marker (nov-142) and mutational sites affecting pyrimidine (pyr-141), purine (pur-102), and histidine (hisG15) biosynthesis in S. aureus strain 8325. The linkage group thus defined is pyr-141-hisG15-nov-142-pur-102-mec-4916. Phage 80alpha previously propagated on a novobiocin-resistant, methicillin-sensitive (Mecs) 8325 strain was used to infect 21 novobiocin-sensitive, methicillin-resistant clinical isolates (including strain DU4916). Among the novobiocin-resistant transductants so obtained from each recipient, between 1 and 5% were methicillin sensitive (reflecting cotransduction of Novr and Mecs). These results are consistent with the genetic determinant of methicillin resistance having a single chromosomal locus in most, if not all, strains of S. aureus.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Kinetics of uptake and incorporation of meso-diaminopimelic acid in different Escherichia coli strains.

The rate at which the peptidoglycan precursor meso-diaminopimelic acid (DAP) is incorporated into the cell wall of Escherichia coli cells was determined by pulse-label experiments. For different E. coli strains, the incorporation rate was compared with the rate of uptake of DAP into the cell. With E. coli W7, a dap lys mutant generally used in this kind of studies, steady-state incorporation was reached only after about 0.75 of the doubling time. This lag period can be ascribed to the presence of a large internal DAP pool in the cells. An E. coli K-12 lysA strain was constructed which could be grown without DAP in its medium. Consequently, due to the higher specific activity of the added [3H]DAP, faster incorporation and higher levels of radioactivity in the peptidoglycan layer were observed in the K-12 lysA strain than in the W7 strain. In addition, uptake and incorporation were faster in steady state (within about 0.2 of the doubling time), indicating a smaller DAP pool. The lag period could be further diminished and the incorporation rate could be increased by feedback inhibition of the biosynthetic pathway to DAP with threonine and methionine. These results make MC4100 lysA a suitable strain for studies on peptidoglycan synthesis. To explain our observations, we suggest the existence of an expandable pool of DAP in E. coli which varies with the DAP concentration in the growth medium. With 2 microgram of DAP per ml, the size of the pool is severalfold the amount of DAP contained in the cell wall. This pool can be partly washed out of the cells. Grown without DAP, MC4100 lysA still has a small pool caused by endogenous synthesis, which accounts for the fact that steady-state [3H]DAP incorporation in the lysA strain still shows a lag period.     

Mutagenesis of anaerobic cultures of Salmonella typhimurium by nitrosoguanidine, diethyl sulfate and 9-aminoacridine

Despite a previous report to the contrary, anaerobic cultures of Salmonella typhimurium strain LT2 hisG46 are revertible (although to a slightly reduced extent) by both N-methyl-N'-nitro-N-nitrosoguanidine and diethyl sulfate, while anaerobic cultures of a strain carrying the frameshift hisC3076 marker are fully revertible by 9-aminoacridine.     

Mutagenic activity and specificity of N-nitrosomethylaniline and N-nitrosodiphenylamine in Salmonella

The carcinogenic nitrosamines, N-nitrosomethylaniline (NMA) and N-nitrosodiphenylamine (NDphA), which have been previously reported negative or very weakly mutagenic in the Salmonella/microsome assay, were found to be mutagenic in the hisG428 Salmonella strain, TA104. NMA was moderately potent and NDphA was about 10% as potent. Mutagenesis by both compounds was dependent on the uvrB mutation and enhanced in strains harboring the plasmid, pKM101. The mutational specificities of NMA and NDphA for base-pair substitutions were determined by assaying their activities in several mutants which are reverted by a limited number, or a single type of base-pair substitution mutation, and additionally by subclassification of revertants. NMA induced predominantly AT----CG transversions and NDphA induced AT----TA transversions. The specificity of NMA and NDphA for mutagenesis at AT base pairs and the lack of sensitivity of the previously employed hisG46 strains for these base changes may be the reason for the previous reports on the lack of mutagenic activity of these compounds. This specificity is quite unusual for nitrosamines and is consistent with the hypothesis that NMA and NDphA lead to DNA damage of different nature than that produced by other nitrosamines.     

Mutations in Salmonella typhimurium recovered from livers and spleens of mice

Balb/c mice were inoculated intraperitoneally with TA2662, a smooth derivative of the Salmonella typhimurium Ames tester strain (TA102) which carries the mutable hisG locus on a multicopy plasmid, or TA103, which carries the same hisG gene on the chromosome. The bacteria were recovered at various times from the livers and spleens of the infected mice. Total numbers of bacteria were determined and the mutant frequency was estimated. The frequency of occurrence of histidine prototrophs in experiments using TA2662 was substantially above the frequency found with this strain grown in vitro. The mutant frequencies in experiments using TA103 recovered from mice were also highly significantly increased above background. We did not identify factors which might suggest selection in vivo for histidine prototrophs. There is sufficient histidine in body fluids of the host for the growth of His- bacteria. The His- and His+ derivatives were found to grow equally well in vitro in the presence of amounts of histidine approximating concentrations known to exist in vivo. It is probable that mutations in TA2662 are greatly underestimated, since the hisG-containing plasmid is lost at relatively high frequency during incubation in a variety of conditions.     

Isolation of a mutant of Salmonella typhimurium strain TA1535 with decreased levels of glutathione (GSH-). Primary characterization and chemical mutagenesis studies

A mutant of Salmonella typhimurium strain TA1535 with decreased glutathione (GSH) levels was isolated after treatment with UV and selection for N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) resistance; this GSH- mutant also exhibited increased resistance to MNNG, the methyl analog of ENNG. Estimation of the cellular GSH content showed that the GSH- derivative contained about 20% of the GSH levels found in TA1535. In mutagenicity tests (hisG46 leads to His+), the GSH- strain required the presence of GSH or L-cysteine in the medium for an optimal phenotypic expression and/or growth of spontaneous and induced His+ revertants, and may, therefore, be allelic to cys mutants of Salmonella described earlier. The mutagenic activity of MNNG, ENNG and 1,2-dibromoethane (DBE), but not that of N-ethylnitrosourea (ENU), was strongly reduced in TA1535/GSH-; pretreatment of the strain with GSH restored the mutagenicity of the first 3 chemicals to levels normally found in TA1535. The results support the current view that MNNG, ENNG and DBE, but not ENU, can be activated via reaction with GSH to species of higher reactivity and mutagenicity. It is concluded that the present GSH- strain can be used to study more systematically the role of GSH in the bioactivation and -deactivation of xenobiotics to mutagenic factors.     

Indications that mutagenesis in Salmonella may be subject to catabolite repression

Spontaneous reversion of the base-pair substitution trpE8 marker in the LT2 sub-line of Salmonella typhimurium is significantly increased in the presence of the ultraviolet light-protecting and mutation-enhancing plasmid pKM101. The numbers of Trp⁺ revertants arising on plates of defined medium supplemented with trace amounts of nutrient broth have been found to depend upon the nature of the carbon source provided to support growth of both the background lawn and any revertants which may arise. For example, the yield of Trp⁺ revertants can be some 5–8 times greater when glycerol is the carbon source as compared to when glucose is the carbon source. S. typhimurium strain TA100, which carries the base-pair substitution hisG46 marker and pKM101, shows a similar response, although the difference is much smaller. Time-course experiments using both carbon sources indicate that the final trpE8 → Trp⁺ mutation yield is depressed by glucose rather than enhanced by a ‘mutagenic’ effect of glycerol. These results are consistent with the idea that a glucose-repressible function responsible for generating mutations can be switched on by growth on glycerol as sole carbon source. Evidence is also presented that many more mutational events occur in response to a mild temperature stress (42°) in populations growing on glycerol as carbon source than occur in populations growing on glucose.     

Phenotypic and reversion analysis of a Salmonella typhimurium constructed to have an arginine codon at the hisG46 missense codon

Of the 6 single-base mutations that would be predicted to change the missense mutation hisG46 away from a proline codon in the Salmonella/microsome mutagen selection assay for histidine-independent revertants, only 5 have been observed. We have used site-specific mutagenesis to make the unobserved mutant [CCC (proline)----CGC (arginine)] codon in the Salmonella genome. Experiments with this arginine mutant demonstrate that, like bacteria containing the hisG46 mutation, bacteria with the arginine missense mutation are histidine auxotrophs which are capable of reversion to histidine independence. However, unlike the ATP phosphoribosyltransferase coded by the hisG46 his G gene (with a proline), the arginine mutant enzyme is partially active. This is indicated by a histidine-independent phenotype when the arginine hisG gene is present in multiple copies.     

Sequence Analysis of Mutations Arising during Prolonged Starvation of Salmonella Typhimurium

We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA). Both of these mutants can revert to His+ via intragenic and extragenic mechanisms. Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site. Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites. Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs. A/T sites as well as on intragenic sites vs. extragenic suppressor sites. Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants. Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations. The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium. Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.