Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes

Mutation Research, 301 (1993) 7-12 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-7992/93/$06.00

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MUTLET 00739

Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes Masahiko Watanabe, Toshio Sofuni and Takehiko Nohmi Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan (Received 8 April 1992) (Revision received 4 August 1992) (Accepted 6 August 1992)

Keywords: Aromatic amines; Nitroarenes; Acetyltransferase; pKM101

Summary Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamine O-acetyltransferase (OAT) activity. We have demonstrated that this strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes. In this paper, we compared the sensitivities of YG1024 with those of S. typhimurium YG1012, which has about 4 times higher OAT activity than YG1024 but lacks plasmid pKM101. It turned out that YG1024 was more sensitive to the mutagenic actions of 1-aminonaphthalene, 1-nitropyrene, 1,8-dinitropyrene and 2-nitronaphthalene than YG1012 and showed comparable sensitivity to 2-hydroxyacetylaminofluorene, 2-aminoanthracene and 2-amino-6-methyldipyrido[1,2-a : 3',2'-d]imidazole (GIu-P-1) to YG1012. These results suggested that YG1024 is more suitable than YG1012 for the efficient detection of mutagenic aromatic amines and nitroarenes.

We have developed Salmonella typhimurium YG1024, a new derivative of Ames tester strain TA98, with a high level of N-hydroxyarylamine O-acetyltransferase (OAT) activity (Watanabe et al., 1990). This strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes (Einist6 et al., 1991). This strain is also sensitive to complex mixtures containing mutagenic aromatic amines a n d / o r nitroarenes: smokers' urine Correspondence: Dr. T. Nohmi, Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan.

specimens (Einist/5 et al., 1990) and the mutagens recovered from municipal river water (Sayato et al., 1990) were more mutagenic to YG1024 than to TA98. During construction of the strain, we have developed a subcloned plasmid pYG213. Plasmid pYG213 could not be introduced into the pKM101-bearing strains TA98 and TA100 since the antibiotic resistance marker of pYG213, ampicillin, is the same as that of pKM101. However, the plasmid produced, an extremely high level of OAT activity and the strains harboring pYG213 showed about 4 times higher OAT activity than did YG1024 (Watanabe et al., 1990). Thus, it was suggested that the strain harboring

pYG213 might be more useful than YG1024 for the efficient detection of mutagenic amino- or nitro-aromatics although the strain does not harbor plasmid pKM101. In fact, YG1012, a derivative of T A 1 5 3 8 / 1 , 8 - D N P harboring pYG213, shows slightly higher sensitivity to the mutagenic action of benzidine with hamster $9 activation than does YG1024 (Josephy, 1989). In order to determine which strain is m o r e suitable for the general detection of environmental aromatic amines and nitroarenes, we compared the sensitivities of YG1024 with those of YG1012 using eight hydroxyamino-, amino- and nitro-aromatic compounds. The results suggested that the presence of plasmid pKM101 is required for the efficient detection of some frameshift mutagens and thus YG1024 is more useful than YG1012 for the sensitive detection of mutagenic aromatic amines and nitroarenes in the environment. Materials and methods Chemicals and bacterial strains 1-Nitropyrene (1-NP, CAS No. 5522-43-0) was purchased from Tokyo Kasei Kogyo (Tokyo) and was further purified by high-performance liquid chromatography as described (Watanabe et al., 1989). 1,8-Dinitropyrene (1,8-DNP, CAS No. 42397-65-9) was obtained from Dr. N. Miyata, National Institute of Hygienic Sciences, Tokyo. 2-Hydroxyacetylaminofluorene (2-hydroxy-AAF, CAS No. 53-95-2) was purchased from Chemsyn Science Laboratories (Lenexa, KS). 1-Aminonaphthalene (I-AN, CAS No. 134-32-7) was provided by the Ministry of Health and Welfare, Japan. 2-Nitronaphthalene (2-NN, CAS No. 58189-5) was purchased from Aldrich (Milwaukee, WI). 2-Amino-6-methyldipyrido[1,2-a : 3',2'-d]imidazole (Glu-P-1, CAS No. 67730-11-4), 2-aminoanthracene (2-AA, CAS No. 613-13-8) and 4-nitroquinoline 1-oxide (4-NQO, CAS No. 56-57-5) were purchased from W a k o Pure Chemical Industries (Osaka). T h e bacterial strains used in this study are described in Table 1. Mutagenicity assay The Salmonella mutagenicity assay was carried out according to the method of Maron and A m e s (1983) combined with a 'preincubation proce-

TABLE 1 BACTERIAL STRAINS AND THEIR O-ACETYLTRANSFERASE ACTIVITIES Strain

Description

plasmid pKM101 YG1012 TA1538/1,8-DNP (pYG213) YG1019 TA1538/1,8-DNP (pYG219) YG1020 TA98 (pBR322-Aps) + YG1024 TA98 (pYG219) +

OAT activity a 228.0 54.6 1.0 47.4

All strains in Table 1 are derived from Salmonella typhimurium LT2, and have genotypes of A (gal, chl, uvrB, b/o), rfa, hisD3052 (Maron and Ames, 1983). TA1538/1,8-DNP was derived from TA98/1,8-DNP6 by curing plasmid pKM101 (Watanabe et al., 1989) and has no activities of isoniazid- and 2-aminofluorene-N-acetyltransferaseand N-hydroxy-Glu-P-1O-acetyltransferase (Watanabe et al., 1990). TA98/1,8-DNP6 was isolated as a 1,8-dinitropyrene-resistant strain from TA98 (McCoy et al., 1981) and has a frameshift mutation in the gene encoding OAT (Watanabe et al., 1992). pYG213 and pYG219 are derivatives of plasmid pBR322 carrying the OAT gene at different sites (Watanabe et al., 1990). a OAT activity was measured using N-hydroxy-Giu-P-1as a substrate. The reaction mixture containing N-hydroxy-GluP-1 (0.02 mM), acetyl-CoA (1 mM), dithiothreitol (1 mM), Tris-HCl buffer, pH 7.5 (50 raM) and cytosol was incubated for 5 min at 37°C, and the reaction was terminated by adding 600 ttl methanol. After centrifugation, the fluorescence intensity at 445 nm with excitation wavelength at 376 nm was measured. The units of enzyme activity is nmole/min/mg protein.

dure'. Phenobarbital- and 5,6-benzoflavone-induced rat liver $9 was purchased from Kikkoman Co. (Chiba, Japan), and the cofactors were purchased from Oriental Yeast Co., Ltd. (Tokyo). The $9 fractions combined with cofactors were used for the metabolic activation of 1-AN, GIu-P1 and 2-AA. Oxoid nutrient broth No. 2 was used for overnight culture. Ampicillin (25 / z g / m l ) a n d / o r tetracycline (6.25 / z g / m l ) was added to the broth if necessary. The experiments were repeated at least twice to confirm that the relative sensitivity of the strains is reproducible. Mean values of His + revertants per plate are indicated in Fig. 1 and Table 2. Results and discussion S. typhimurium YG1012, the derivative of strain T A 1 5 3 8 / 1 , 8 - D N P containing plasmid pYG213, has an extremely high O A T activity: the activity

TABLE 2 SENSITIVITIES OF YG1020, YG1024 AND YG1012 TO TYPICAL ARYLHYDROXYLAMINES, AROMATIC AMINES AND NITRO-AROMATIC COMPOUNDS Chemical

2-Hydroxyacetylaminofluorene

Group a

A

Dose (p.g/plate) 0 0.1 0.3 1 3 10

Induced revertants/nmole GIu-P-1 + $9

A

0 0.0003 0.001 0.003 0.01 0.03

Induced revertants/nmole 2-Aminoanthracene + $9

A

0 0.01 0.03 0.1 0.3 1 3

Induced revertants/nmole 1-Aminonaphthalene + $9

B

0 3 10 30 100 300

Induced revertants/nmole 1-Nitropyrene

B

0 0.03 0.1 0.3 1 3

Induced revertants/nmole 1,8-Dinitropyrene

Induced revertants/nmole

B

0 0.0001 0.0003 0.001 0.003 0.01

Revertants per plate YG1020 42 37 41 103 180 587 13 (1.0) 45 60 97

YG1024

YG1012

79

74

203

236

424 1 118 2413 4090 297 (22.8)

557 1433 2805 4076 388 (29.7)

YG1019 71 127 238

594 1310 3179 133 (10.2)

173

123

488

306

358 819 8090 (1.0)

176 313 746 1507 4 233 8 586 144000 (17.8)

1189 2 877 6 692 10 491 266000 (32.9)

636 1664 4018 8 039 154000 (19.0)

45 48 68 80 228 1146 5495 351 (1.0)

176 378 943 3 313 7 329 2716 198 6060 (17.3)

173 725 1842 3 407 285 141 156 10800 (30.7)

123 233 847 1827 3 976 348 66 4660 (13.3)

145 263

149 188 235

124 148 165

535 741 528 3.52 (88.0)

372

249

296 339 1.06 (26.5)

158 158 0.60 (15.0)

97 142

178 387 1213 1953 373 (1.0)

79 348 1307 2975 7071 9 240 3 040 (8.2)

724 1572 65 576 (1.5)

37 84 183 737 2 408 6 246 231000 (1.0)

63 1506 4 971 12102 20 078 23 353 4 780 000 (20.7)

55 264 1497 3 350 797 312 1400 000 (6.1)

141

43 59 44 51 68 72 0.04 (1.0) 27 61

391

330

27 67 79 131 277 1677 395 (1.1) 58 147 475 2 826 4 827 108 809 000 (3.5)

10 TABLE 2 (continued) Chemical

2-Nitronaphthalene

Group a

B

Dose (/~g/plate) 0 3 10 30 100

Induced revertants/nmole

4-Nitroquinoline 1-oxide

C

0 0.03 0.1 0.3 1

Induced revertants/nmole

Revertants per plate YG1020

YG1024

YG1012

YG1019

34 57 199 338 776 2.9 (1.0)

53 154 798 1439 2909 12.9 (4.4)

67 121 293 178 34 3.9 (1.3)

35 64 197 363 208 2.8 (1.0)

34 84 171 560 1276 333 (1.0)

53 112 232 597 1238 345 (1.0)

67 77 133 201 347 85 (0.3)

35 65 87 218 463 116 (0.3)

The number of induced His + revertants per nmole of a test substance was calculated at every dose and the highest value for each chemical and strain is indicated. Numbers printed bold were used for this calculation. The numbers in parentheses represent the values relative to the number of His +-induced revertants per nmole in YG1020. a The chemicals were classified into three groups based on their mutagenicity to the strains. For details see text.

was more than 200 times higher than that of YG1020, a derivative of strain TA98 containing plasmid pBR322-Ap s (Table 1). The level of activity was even about 4 times higher than that of YG1024, which is a derivative of TA98 containing pYG219, another OAT-overproducing plasmid. Both strains YG1020 and YG1024 have plasmid

pKM101 while YG1012 does not. In order to assess the general usefulness of YG1024, we have compared the sensitivities of YG1024 toward eight mutagens with those of YG1020 and YG1012. We also compared the sensitivities of YG1024 with those of YG1019, which lacks pKM101, to evaluate the importance of pKM101 for detecting

2-HydroxyacetylarninoU uorene GIu-P-1 + $9 2-Aminoanthracene + $9 1-Aminonaphthalene + S9 1 -Nitropyrene

7.7

1,8-Dlnltropyrena 2-Nitronaphthalene 4-Nitroquinoline 1-oxide 5

10

Induced His + revertants of YG1024 per nmole Induced His + revertants of YG1019 per nmole

Fig. I. Ratio of induced His + revertants of YG1024 per nmole to those of YG1019 per nmole. YG1024 is TA98 (pYG219), YGI019 is TA1538/1,8-DNP(pYG219). Cross-hatched bars are these of Group A compounds; hatched bars those of Group B compounds.

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the mutagens. The genetic difference between YG1019 and YG1024 with respect to the 1,8DNP-resistant mutation is rendered irrelevant by the presence of pYG219. Based on the results of the mutagenicity tests, we classified the eight chemicals into three groups as follows. Group A includes 2-hydroxy-AAF, GIu-P-1 and 2-AA (Table 2). YG1024 showed a comparable or slightly lower sensitivity to these mutagens than did YG1012. There was little difference between YG1024 and YG1019 with these compounds (Fig. 1), suggesting that these compounds are not strongly dependent on the presence of pKM101 for exerting maximum mutagenicity. Group B includes 1-AN, 1-NP, 1,8-DNP and 2-NN (Table 2). YG1024 showed a 3-5 times higher sensitivity to these chemicals than did YG1012. YG1024 was about 8 and 4 times more sensitive to 1-NP and 2-NN, respectively, than YG1020, while YG1012 showed almost the same level of sensitivity as did YG1020. All the chemicals in group B were more than 4 times more mutagenic to YG1024 than to YG1019 (Fig. 1), suggesting that the chemicals require the presence of pKM101 to exert maximum mutagenicity. One compound, 4-NQO, behaved very differently from the chemicals in groups A and B, in that YG1024 showed almost the same level of sensitivity to this compound as did YG1020. YG1012 showed a lower sensitivity than did YG1024 and YG1020. OAT is not involved in the activation of 4-NQO (Einist6 et al., 1991). Thus, it is reasonable that neither YG1024 nor YG1012 showed a higher sensitivity to this chemical than did YG1020. In previous papers, we have demonstrated that the OAT activity is a rate-limiting step for the metabolic activation of N-hydroxyarylamines derived from nitroarenes and aromatic amines in S. typhimurium. In fact, YG1024 and YG1029, the OAT-overproducing derivatives of TA98 and TA100, respectively, were highly sensitive to the mutagenic actions of N-hydroxyarylamines, aromatic amines and nitroarenes. We expected, therefore, that the highest OAT-producing strain, YG1012, might be more sensitive to the chemicals than YG1024, although YG1012 does not have plasmid pKM101. As shown in Table 2, however, this assumption proved to be incorrect:

YG1012 showed 3-5 times lower sensitivity to the chemicals classified in group B than did YG1024. The reason for the lower sensitivity of YG1012 is the lack of plasmid pKM101 since the chemicals of group B required pKM101 for their maximum mutagenicity as indicated by a comparison of strains YG1024 and YG1019 (Fig. 1). On the other hand, YG1012 did not show significantly higher sensitivities to the chemicals classified in group A than did YG1024 (Table 2) although these chemicals did not require plasmid pKM101 for their mutagenesis (Fig. 1). The level of OAT activity in YG1024 seems to be high enough for the intracellular metabolic activation of N-hydroxyarylamines. Plasmid pKM101 carries mucAB genes, which are involved in the error-prone repair of Escherichia coli and S. typhimurium (Walker, 1984). It is known that some base-change type mutagens such as furylfuramide, methyl methanesulfonate and aflatoxin B~, are heavily dependent on pKM101 to exert their mutagenicities (McCann et al., 1975). Our results shown in Fig. 1 suggest that not only the base-change type mutagens but also some frameshift mutagens such as 1-NP and 1,8-DNP (Watanabe et al., 1989; Bell et al., 1991) require the presence of pKM101 to exert maximum mutagenicity. In this paper, we report that plasmid pKM101 is required for the efficient detection of some frameshift mutagens and thus YG1024 is more useful than YG1012 for the sensitive detection of mutagenic aromatic amines and nitroarenes.

Acknowledgements We thank Dr. P.D. Josephy for reviewing the manuscript. This work was supported by a grantin-aid from the Japan Health Sciences Foundation.

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Communicated by F.H. Sobels