Mutagenesis, Vol. 15, No. 6, 495-502,
November 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Comparison of the sensitivities of Salmonella typhimurium strains TA102 and TA2638A to 16 mutagens
Pharmacia & Upjohn AB, Plant Plasma and Refacto, S-112 87, Stockholm and 1 AstraZeneca R&D Södertälje, Safety Assessment, S-151 85 Södertälje, Sweden
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Abstract |
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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.
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Introduction |
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The standard set of bacterial strains used in the mutagenicity testing of chemicals and pharmaceuticals comprises five strains: Salmonella typhimurium TA1537, TA1535, TA100, TA98 and TA102 or Escherichia coli WP2 uvrA(pkM101) or WP2 (pKM101) (Sofuni, 1994
; OECD, 1997). Strain TA102 and the E.coli strains have an AT base pair at the primary reversion site and are included in the set to detect oxidative compounds, cross-linking agents and hydrazines (OECD, 1997). It has been shown that the excision repair deficient strains carrying a deletion in the uvrA gene are less sensitive to cross-linking agents (Wilcox et al., 1990). Therefore, in cases where the detection of cross-linking agents is desired, the use of either the Salmonella strain TA102, which is repair proficient, or of both a DNA repair-deficient and a DNA repair-proficient E. coli strain is recommended (Gatehouse et al., 1994; ICH, 1994; OECD, 1997).
TA102 can be used to replace both types of E.coli, but has disadvantages, namely a propensity to form small colonies and a high incidence of spontaneous revertant colonies. The former renders automatic counting less accurate (Wilcox et al., 1993) while the latter leads to a reduced sensitivity (Mahon et al., 1989). Additional uncertainty regarding the interpretation of results with strain TA102 arises from the observations that non-mutagenic mechanisms may lead to increased numbers of revertant colonies (Albertini and Gocke, 1988; Gocke, 1989).
S.typhimurium TA2638a and TA102 both contain an ochre mutation in the gene hisG428 and are DNA repair proficient. Both strains contain a deep rough mutation, increasing the permeability of the cells to large molecules, and contain the plasmid pKM101 which confers an increase in error-prone DNA repair. The mutant hisG428 gene occurs in the chromosomal DNA of strain TA2638 but is carried on the multicopy plasmid pAQ1 in strain TA102 (Levin et al., 1982).
The aim of the present study was to investigate the possibility of using TA2638a, a derivative of TA2638 with improved growth characteristics (C.M.Wehr, personal communication), in place of TA102 for routine testing and to see if the derived strain gave the same results as obtained previously with strain TA2638. To this end, the sensitivity of strains TA2638a and TA102 to 16 compounds that are preferentially mutagenic to strain TA102 was examined (Levin et al., 1982; De Flora et al., 1984; Wilcox et al., 1993). An additional aim was to compare the spontaneous reversion frequency and colony size of the two strains.
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Materials and methods |
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Chemicals and biologicals
All chemicals used for testing were of the highest purity available. The chemical sources, solvents used and CAS numbers based on chemical selection are shown in Table I
. The same lot of each chemical, except streptonigrin and mitomycin C, was used for all experiments.
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Liver homogenate (protein content 36.5 mg/ml) was purchased from Molecular Toxicology, Inc. (Annapolis MD, USA). It was prepared from the livers of Sprague–Dawley male rats, induced by Aroclor 1254 and stored at –70°C. The S9 mix was prepared with proportions of S9 fraction, co-factors and buffers according to Ames et al. (1975).
S.typhimurium strains TA102 and TA2638a were kindly provided by Professor B.N.Ames (University of California, Berkeley, CA, USA). The suffix `a' designates a reconstruction of strain TA2638 to improve growth characteristics (C.M.Wehr, personal communication). The genetic characteristics of each strain were tested as recommended (Maron and Ames, 1983).
Performance of the test
The Ames test was performed as described by Ames et al. (1975) using the plate incorporation method, except for formaldehyde, which was tested by pre-incubation. Each bacterial strain was grown in nutrient broth, supplemented with 2 µg/ml tetracycline and 25 µg/ml ampicillin for TA102 or with 25 µg/ml of ampicillin for TA2638a. One hundred microlitres of bacterial culture was added to 2.0 ml of 45°C molten top agar, containing histidine and trace amounts of biotin, 0.1 ml of the test compound or solvent and 0.5 ml of sodium phosphate buffer (pH 7.4) or S9 mix. The mixture was poured on to minimal glucose agar plates (25 ml/plate) and incubated for 72 h at 37°C; revertant colonies were then counted by an automatic colony counter (Perceptive Instruments Ltd, Halstead, Essex, UK).
All compounds were tested twice with triplicate plates for all doses and solvent controls. A dose of 5 mg/plate was used as the highest dose if no toxicity was observed. Positive controls were included in each experiment. Phenylglyoxal was used in strains TA102 and TA2638a without metabolic activation at a dose of 500 µg/plate. 2-Aminoanthracene was used at a dose of 20 µg/plate for TA102 and TA2638a with metabolic activation.
Statistical analysis
Results were analysed by statistical methods recommended by Mahon et al. (1989). Differences in the number of revertants on the treated plates and the solvent controls were tested for significance on the square-root transformed mean colony counts, using a one-tailed t-test with correction for multiple comparisons according to Dunnett (1955). The analysis was performed using the SAS statistical analysis system (SAS, NC, USA). Results were considered to be significant when P was <0.01.
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Results |
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The experience in our laboratory from working with these two strains is that revertant colonies of TA2638a are relatively uniform in size and easily visible by eye while TA102 colonies are of variable size, some of them being too small to count by eye (Figure 1
). The viability after overnight culturing was ~4 x 109 bacteria/ml for strain TA2638a and 3 x 109 bacteria/ml for strain TA102 as determined by dilution plating. The spontaneous mutatant frequency of strain TA102 was both higher and more variable than that of TA2638a (Table II). The variability in the number of TA102 revertants in the solvent control necessitated repeating tests with this strain. The number of revertants in the solvent controls for TA102 was within the range found previously (Levin et al., 1982; Wilcox et al., 1993). TA2638a showed a higher spontaneous mutation frequency than found in the literature for TA2638, which is approximately 30–50 (Watanabe et al., 1996).
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The test results for the 16 compounds are summarized in Table I
together with a quantitative comparison of the sensitivities of the two bacterial strains. The data from the tests are shown in Appendix I, together with the fold increases in revertants at each dose. The result was classified as positive (`+' in Table II) when there were significantly (P 
0.01) more revertant colonies on the treated plates than on the solvent control plates. To compare the sensitivity of the strains for each test compound, a criterion for sensitivity was established. If there was a significance (P 0.01) in at least two lower doses in one strain, that strain was considered to be the more sensitive one (`++' in Table I) for that particular compound. If the maximum magnitude in the response to a test compound (expressed as fold increase over the solvent control) was at least twice the fold increase for the other strain, this is marked with a `c' in Table I.
Bleomycin sulfate and streptomycin sulfate produced different responses in the two strains. TA2638a did not show a statistically significant increase, although a 10-fold higher dose was used for bleomycin sulphate and a >7-fold higher dose for streptomycin sulphate. In strain TA102, significant increases in the number of revertants were seen with bleomycin sulfate (at 
0.05 µg/plate) and with streptomycin (at 3.3 µg/plate).
2-Butanone peroxide gave statistically significant effects at 104 µg/plate in test 1 with both TA102 and TA2638a. In test 2, the statistically significant increase was only seen in TA2638a at 195 µg/plate although an increase of 1.5-fold was seen in TA102 at a lower dose. The compound was more toxic to TA102.
t-Butyl hydroperoxide produced a statistically significant increase in the number of revertants at 45 µg/plate. The maximum fold increase in TA2638a (14.1) was twice as high as that in TA102 (6.8).
Cumene hydroperoxide produced a statistically significant increase in the number of revertants at 33 µg/plate in TA102 and at 50 µg/plate in TA2638a. The maximum fold increase in TA2638a (8.7) was twice as high as that in TA102 (3.9).
Danthron caused a significant increase in the number of TA2638a and TA102 revertants, with metabolic activation, from 10 µg/plate. The maximum fold increase was ~2–3 for both strains.
Formaldehyde caused a statistically significant increase in the number of revertant colonies over the solvent controls in strains TA2638a and TA102 at 17 µg/plate using the pre-incubation method. The maximum fold increase was 3 in strain TA2638a and 2.5 in TA102.
Glutaraldehyde produced a statistically significant increase for both strains at 25 µg/plate. The maximum fold increase was ~4.5 for both strains.
Glyoxal was tested three times, because discordant results were obtained in the first and second tests. A statistically significant increase in the number of revertants was seen in TA102 and TA2638a at 2480 µg/plate in the first test. In the second test, a statistically significant increase was seen at 956 µg/plate in TA2638a, but no significant increase was detected in TA102 although there were twice as many revertant colonies than in the solvent controls. In the third test a statistically significant increase was seen at 944 µg/plate in TA2638a and at 3770 µg/plate in TA102. The maximum fold increase was 5.1 in TA2638a and 2.0 in TA102.
Hydrogen peroxide at 51 µg/plate caused a statistically significant increase in revertants in both tests in TA102. In TA2638a a significant increase was seen at 102 µg/plate. The maximum magnitude of the increase in the two studies in TA102 and TA2638a was 6.0- and 2.7-fold, respectively.
Kethoxal produced a statistically significant increase at the same dose in both TA102 and TA2638a, i.e. at 469 µg/plate. The number of revertants in TA2638a reached a maximum of a 7.1-fold increase while TA102 reached a 3.9-fold increase.
Malondialdehyde produced statistically significant increases in the number of TA102 and TA2638a revertants at 1920 µg/plate and 962 µg/plate, respectively. The fold increase was ~2 in both strains.
Mitomycin C gave a statistically significant increase in the mutagenic activity in TA102 at 0.03 µg/plate while TA2638a was mutagenic first at a 10-fold higher dose. The maximum fold increase in response in TA102 and TA2638 was 5.1 and 2.0, respectively.
Phenylhydrazine–HCl showed a clear statistically significant increase, with metabolic activation, in test 1 in TA102 at 252 µg/plate and in TA2638a at 50 µg/plate. The magnitude of the response was ~2-fold. In the second test, a statistically significant increase was found in TA102 at a single dose at maximal fold increase of 1.5. At the maximum dose (1970 µg/plate), TA2638a showed a statistically significant increase while a toxic effect was seen in TA102.
Sodium dichromate dihydrate produced similar responses in both strains. A statistically significant increase in the number of revertants was seen at 5 µg/plate. Approximately 4-fold increases were seen in both strains.
Streptonigrin produced increases in the number of revertants at similar lowest doses in TA102 and TA2638a. The maximum response was also similar, ~6.0-fold, in both strains.
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Discussion |
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Of the 16 compounds tested, all gave positive results in TA102 while 14 were positive in TA2638a. Those generating negative results were streptomycin sulfate and bleomycin sulfate. Streptomycin was mutagenic to strain TA102 in the study of De Flora et al. (1984). Bleomycin was mutagenic to strain TA102 in the studies of Levin et al. (1982), Wilcox et al. (1993) and Watanabe et al. (1995), but variable results were seen in E.coli WP2 (Wilcox et al., 1993
; Watanabe et al., 1995). Kanamycin and streptomycin have been shown to increase the number of revertant colonies in TA102 bacteria plated in the absence of histidine (Gocke, 1989). This effect was ascribed to a misreading of the mutated site in the histidine gene allowing some cells to grow in the absence of histidine. No increase in the number of revertant colonies was seen in strains TA100 or TA104. These findings may explain the different results obtained with streptomycin in TA2638a and TA102. In the case of bleomycin, it has been found that this compound induces deletions in the HisG428 gene when this is located on the PAQ1 plasmid but not when the gene is located on the chromosome, which may explain the different sensitivities of the two strains (Levin et al., 1984). The two strains were considered equally sensitive with respect to lowest effective dose and maximum fold increase in seven of the 16 cases, i.e. danthron, formaldehyde, glutaraldehyde, malondialdehyde, phenylhydrazine–HCl, sodium dichromate dihydrate and streptonigrin. The lowest effective dose was the same in both strains while the fold increase was higher for strain TA2638a than for TA102 for four compounds: kethoxal, cumene hydroperoxide, t-butyl hydroperoxide and glyoxal.
Strain TA102 responded at a lower concentration than TA2638a to hydrogen peroxide and mitomycin C, while TA2638a was more sensitive to 2-butanone peroxide and glyoxal.
Levin et al. (1982) compared the sensitivity of strains TA102 and TA2638 to 18 compounds and treatment with UV and X-ray irradiation. Discordant results were obtained for bleomycin and malondialdehyde for the two strains. Negative results were obtained with formaldehyde in both strains. It was concluded that strain TA2638 was generally less sensitive than TA102. This was based on a lower number of induced revertants in TA2638. However, the results also show that the fold increase in the number of revertants was lower in TA102 than in TA2638.
Comparing the results of the present study with those of Levy et al., positive results were seen with formaldehyde and malondialdehyde. Formaldehyde was tested using the liquid pre-incubation method in the present study. The compound has been shown to be detected more readily using the liquid pre-incubation method (). The present results agreed qualitatively with those of Levin et al. (1982) for the other 10 compounds that were tested in both studies.
Watanabe et al. (1995, 1996, 1998a,b) performed collaborative trials with TA102, TA2638 and E.coli strains WP2/pKM101 and WP2/pKM101. The results were in agreement in 73 of 81 (90%) cases for the two strains. For the compounds classified as oxidative or cross-linking agents, there was agreement in 25 of 28 cases. The sensitivity of the two strains was similar as judged by the lowest effective dose. The results for the 11 oxidative and cross-linking agents that were tested in the collaborative studies of Watanabe et al. (1998b) and in the present study were in agreement.
The results obtained in this study with strain TA2638a are in good agreement with those obtained in previous studies with TA2638. The present and previous studies reveal a close concordance for results for strains TA2638 and TA2638a on the one hand and TA102 on the other. The results of the present study do not indicate a difference in the sensitivity of the two strains. TA2638a should detect weak mutagens more reliably than TA102 considering the lower spontaneous revertant frequency. The absence of small colonies in TA2638a will result in more accurate colony counting when using automatic colony counters. Taking these observations together, it is concluded that TA2638a should be considered as an alternative to strain TA102 in the standard set of strains used in the Ames test.
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Notes |
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* To whom correspondence should be addressed. E-mail: george.bolcsfoldi{at}astrazeneca.com
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References |
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Received on March 31, 2000; accepted on August 15, 2000.
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