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Mutagenicity of some congeners of benzidine in the salmonella typhimurium assay system
Cancer Letters, 4 (1977) 21--25 © Elsevier/North-Holland Scientific Publishers Ltd.
21
MUTAGENICITY OF SOME CONGENERS OF BENZIDINE IN THE SALMONELLA TYPHIMURIUM ASSAY SYSTEM
EDWARD J. LAZEAR and SHIRLEY C. LOUIE
Division of Mutagenesis Research, National Center for Toxicological Research, Jefferson, Arkansas 72079 (U.S.A.) (Received 1 August 1977) (Revised version received 26 September 1977) (Accepted 3 October 1977)
SUMMARY
The analogs of benzidine were assayed for mutagenicity using Salmonella typhimurium TA-98 and TA-100 and a mouse liver enzyme preparation. Only 4-aminobiphenyl produced b o t h frameshift and base pair substitution mutations and 3,3'-dichlorobenzidine was the only c o m p o u n d which was mutagenic w i t h o u t the mammalian enzyme factor. When hydrochloride salts of the parent c o m p o u n d s were made to improve their stability for animal feeding experiments, the mutagenicity for the Salmonella tester strains was reduced except for 3,3'-dimethylbenzidine. Animal feeding trials in mice are underway to determine the dose response relationship of t u m o r incidence and molecular configuration.
INTRODUCTION
Benzidine and some of its congeners are commercially important c o m p o u n d s in the chemical industry and millions of pounds of these chemicals are produced each year. The fact that some of these compounds have been found to induce cancer in man and animals has p r o m p t e d a concerted effort to find less hazardous substitutes. The search for these substitute compounds is impeded by the fact that in vivo tests in experimental animals are expensive, require large numbers of animals and m a y cover several years from initiation to final analysis. Ames et al. [1,2,10] have constructed a number of strains of S. typhimurium which have been used extensively to detect carcinogens as mu~agens. These in vitro microbiological tests are rapid, economical and reproducible. McCann et al. [ 11 ] recently published a summation of results of bacterial mutagenicity tests on 300 chemicals. The relatively high correlation (90%) between the
22
mutagenicity in the Salmonella test system and carcinogenicity in animals suggests that the former test system can be used as a reliable prescreening test for potential carcinogenicity. The merits land inconsistencies in the correlations have been discussed at length elsewhere [3,12]. As an integral part of the National Center for Toxicological Research Tier approach to carcinogenicity testing, the different lots of chemicals that are scheduled to be tested in mice in the near future were tested in the Salmonella assay system to evaluate their relative mutagenic potential. While this work was in progress, Garner et al. [7] reported their results with 6 benzidine analogs using one of the tester strains constructed by Ames. O u r study, which used different tester strains and a different mammalian enzyme system, confirmed the findings of Garner et al. [7] and expanded the study to include other analogs of benzidine not previously reported. MATERIALS AND METHODS
The strains of S. typhimurium namely TA-98 and TA-100 [10] were supplied by Dr. Bruce Ames and the stock cultures were maintained in our laboratory in accordance with the instructions provided with the cultures. The test methods have been described in detail [2] and only 2 minor modifications were used in our laboratory. For this study, the mammalian enzyme preparations (S-9 fractions) were prepared from uninduced adult male BALB/c mice, 15--20 weeks of age, and a Polytron grinder was used to mince the tissue prior to centrifugation. The chemicals were purchased from various suppliers and some analogs were synthesized by personnel at the NCTR or on contract. The 3,3'-dimethoxybenzidine--2HC1, the 3,3'-dichlorobenzidine and its dihydrochloride and the 3,3'-dimethylbenzidine were purchased from Pfaltz and Bauer Company. The benzidine and the 4-aminobiphenyl were o b t a i n e d from Fisher Scientific Company and Aldrich Chemical Company, respectively. The other compounds were synthesized at the Center or on contract. Assays of the c o m p o u n d s as received from the manufacturer or after purification in our laboratory showed negligible amounts of vacuum volatiles. No extraneous GC responses were observed, characteristic analytical properties were confirmed and the salts yielded the proper amounts of free amines. The c o m p o u n d s were considered essentially pure and acceptable for use in animal tests. Specific details on the methods of synthesis, specific properties or m e t h o d s of analysis will be supplied on request [4,5,9]. For the microbiological tests, the c o m p o u n d s were dissolved and diluted in dimethylsulfoxide and added in 0.1 ml amounts to the t o p agar. The plates were then incubated at 37°C for 48 h and counted on an automatic Artek Colony Counter. Each data point was the average of 6 replicate plates. RESULTS AND DISCUSSION Table 1 compares the results of the mutagenicity tests on the congeners of
23 TABLE 1 MUTAGENICITY OF CONGENERS OF BENZIDINE IN THE S A L M O N E L L A ASSAY SYSTEM Compound
Conc ug/plate
Tester strain TA-98
TA-100
Revertants/plate a 4-Aminobiphenyl 4-Aminobiphenyl--HC1 Benzidine Benzidine--2HC1 3,3'-Dimethylbenzidine 3,3'-Dimethylbenzidine--2HC1 3,3~-Dimethoxybenzidine 3,3~-Dimethoxybenzidine--2HCl 3.3r-Dichlorobenzidine 3,3'-Dichlorobenzidine--2HC1
613 1139 455 635 198 356 172 201 14 16 280 360 0 0 12 11 2071 2012 1612 1872
50 100 50 100 50 100 50 100 50 100 50 100 50 100 50 100 50 100 50 100
Av. Spontaneous Mutation Frequency
(320) b (622) (244) (383)
36 Range
(26--42)
1212 1456 1152 1455 57 93 114 91 3 10 64 92 0 0 7 0 62 66 66 70 137 (122--161)
a Average spontaneous mutation frequency has been subtracted from the entries. b Revertants without mammalian enzyme fractions. All of the compounds were tested, but only the positive results were reported.
benzidine with 4-aminobiphenyl. In this table, the average spontaneous mutation frequencies have been subtracted from the reported values. Where comparable data are available, the mutation frequencies reported here were slightly lower than have been reported in some other studies [7,11]. These lower values are probably due to the fact that the uninduced mouse S-9 preparations have a lower enzymatic activity level than the induced rat S-9 fractions used in other studies. The control compounds, the 4-aminobiphenyl analogs, caused both base pair substitution and frameshift mutations as indicated b y their reactivity with both tester strains. The 3,3'-dichlorobenzidine c o m p o u n d s were moderately mutagenic w i t h o u t the mammalian enzyme preparation; however, the muta-
24
genicity was increased markedly when the S-9 fraction was added to the reaction mixtures. Based on the number of revertant colonies as a measure of mutagenicity, the 3,3'-dichlorobenzidine compounds were the most active mutagens,and the 3,3'-dimethoxybenzidine analogs were the least mutagenic. All of the compounds have been reported to be carcinogenic in experimental animals, b u t they have n o t been studied extensively in mice [6,8]. To improve the solubility and stability of the c o m p o u n d s for the long-term, low dose animal feeding experiments, the compounds were dissolved in aqueous HC1 (0.01 N, pH 2). When the mutagenicities of these salts were compared with those of the parent compounds, the hydrochloric acid tended to decrease slightly the revertant colony counts. Only in the case of the 3,3'-dimelthylbenzidine did the mutagenicity increase when the hydrochloric acid was added to the compound. Animal feeding experiments are in progress or are planned to evaluate the carcinogenic potential of these hydrochloride salts of benzidine in mice. Preliminary results with benzidine and 4-aminobiphenyl suggest that there is a dose response relationship for liver neoplasias in all groups except the controls. When additional histopathological data become available, attempts will be made to correlate the in vitro results with the toxicity and carcinogenicity of all the compounds. ACKNOWLEDGEMENTS
We acknowledge the expert technical assistance of J.G. Shaddo~k, P.R. Barren and D.M. Nestorick. REFERENCES 1 Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. (1973) Carcinogens are mutagerm: A simple test system combining liver homogenates for activations and bacteria for detection. Proc. Natl. Acad. Sci. USA, 70, 2281--2285. 2 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31,346--364. 3 Barsch, H. (1976) Predictive value of mutagenicity tests in chemical carcinogenesis. Mutat. Res., 3 8 , 1 7 7 - - 1 9 0 . 4 Bowman, M.C., King, J.R. and Holder, C.L. (1976) Benzidine and congeners: Analytical chemical properties and trace analysis in five substrates. Intern. J. Environ. Anal. Chem., 4, 205--223. 5 Bowman, M.C. and Rushing, L.G. (1977) 3,3'-Dichlorobenzidine: Trace analysis in animal chow, wastewater and human urine b y three gas chromatographic procedures. Arch. Environ. Contain. Toxicol. In press. 6 Clayson, D.B. (1962) Chemical carcinogenesis. Churchill, London. 7 Garner, R.C., Walpole, A.L. and Rose, F.L. (1975) Testing of some benzidine analogues for microsomal activation to bacterial mutagens. Cancer Lett., 1, 39--42. 8 Haley, T.J. (1975) Benzidinerevisited: A r e v i e w o f the literature and problems associated with the use of benzidine and its congeners. Clin. Toxicol., 8, 13--42.
25 9 Holder, C.L., King, J.R. and Bowman, M.C. (1976) 4-Aminobiphenyl, 2-naphthylamine and analogs. Analytical properties and trace analysis in five substrates. J. Toxicol Environ. Health, 2,111--129. 10 McCann, J., Spingarn, N.E., Kobori, J. and Ames, B.N. (1975) The detection of carcino gens as mutagens: Bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Sci. ~SA, 72,979--983. 11 McCann, J., Choi, E., Yamaski, E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test. Assay of 300 chemicals. Proc. Natl. Acad. Sci. USA, 73, 5135--5139. 12 Sivik, A. (1976) The Ames assay. Science, 193,272--274.
21
MUTAGENICITY OF SOME CONGENERS OF BENZIDINE IN THE SALMONELLA TYPHIMURIUM ASSAY SYSTEM
EDWARD J. LAZEAR and SHIRLEY C. LOUIE
Division of Mutagenesis Research, National Center for Toxicological Research, Jefferson, Arkansas 72079 (U.S.A.) (Received 1 August 1977) (Revised version received 26 September 1977) (Accepted 3 October 1977)
SUMMARY
The analogs of benzidine were assayed for mutagenicity using Salmonella typhimurium TA-98 and TA-100 and a mouse liver enzyme preparation. Only 4-aminobiphenyl produced b o t h frameshift and base pair substitution mutations and 3,3'-dichlorobenzidine was the only c o m p o u n d which was mutagenic w i t h o u t the mammalian enzyme factor. When hydrochloride salts of the parent c o m p o u n d s were made to improve their stability for animal feeding experiments, the mutagenicity for the Salmonella tester strains was reduced except for 3,3'-dimethylbenzidine. Animal feeding trials in mice are underway to determine the dose response relationship of t u m o r incidence and molecular configuration.
INTRODUCTION
Benzidine and some of its congeners are commercially important c o m p o u n d s in the chemical industry and millions of pounds of these chemicals are produced each year. The fact that some of these compounds have been found to induce cancer in man and animals has p r o m p t e d a concerted effort to find less hazardous substitutes. The search for these substitute compounds is impeded by the fact that in vivo tests in experimental animals are expensive, require large numbers of animals and m a y cover several years from initiation to final analysis. Ames et al. [1,2,10] have constructed a number of strains of S. typhimurium which have been used extensively to detect carcinogens as mu~agens. These in vitro microbiological tests are rapid, economical and reproducible. McCann et al. [ 11 ] recently published a summation of results of bacterial mutagenicity tests on 300 chemicals. The relatively high correlation (90%) between the
22
mutagenicity in the Salmonella test system and carcinogenicity in animals suggests that the former test system can be used as a reliable prescreening test for potential carcinogenicity. The merits land inconsistencies in the correlations have been discussed at length elsewhere [3,12]. As an integral part of the National Center for Toxicological Research Tier approach to carcinogenicity testing, the different lots of chemicals that are scheduled to be tested in mice in the near future were tested in the Salmonella assay system to evaluate their relative mutagenic potential. While this work was in progress, Garner et al. [7] reported their results with 6 benzidine analogs using one of the tester strains constructed by Ames. O u r study, which used different tester strains and a different mammalian enzyme system, confirmed the findings of Garner et al. [7] and expanded the study to include other analogs of benzidine not previously reported. MATERIALS AND METHODS
The strains of S. typhimurium namely TA-98 and TA-100 [10] were supplied by Dr. Bruce Ames and the stock cultures were maintained in our laboratory in accordance with the instructions provided with the cultures. The test methods have been described in detail [2] and only 2 minor modifications were used in our laboratory. For this study, the mammalian enzyme preparations (S-9 fractions) were prepared from uninduced adult male BALB/c mice, 15--20 weeks of age, and a Polytron grinder was used to mince the tissue prior to centrifugation. The chemicals were purchased from various suppliers and some analogs were synthesized by personnel at the NCTR or on contract. The 3,3'-dimethoxybenzidine--2HC1, the 3,3'-dichlorobenzidine and its dihydrochloride and the 3,3'-dimethylbenzidine were purchased from Pfaltz and Bauer Company. The benzidine and the 4-aminobiphenyl were o b t a i n e d from Fisher Scientific Company and Aldrich Chemical Company, respectively. The other compounds were synthesized at the Center or on contract. Assays of the c o m p o u n d s as received from the manufacturer or after purification in our laboratory showed negligible amounts of vacuum volatiles. No extraneous GC responses were observed, characteristic analytical properties were confirmed and the salts yielded the proper amounts of free amines. The c o m p o u n d s were considered essentially pure and acceptable for use in animal tests. Specific details on the methods of synthesis, specific properties or m e t h o d s of analysis will be supplied on request [4,5,9]. For the microbiological tests, the c o m p o u n d s were dissolved and diluted in dimethylsulfoxide and added in 0.1 ml amounts to the t o p agar. The plates were then incubated at 37°C for 48 h and counted on an automatic Artek Colony Counter. Each data point was the average of 6 replicate plates. RESULTS AND DISCUSSION Table 1 compares the results of the mutagenicity tests on the congeners of
23 TABLE 1 MUTAGENICITY OF CONGENERS OF BENZIDINE IN THE S A L M O N E L L A ASSAY SYSTEM Compound
Conc ug/plate
Tester strain TA-98
TA-100
Revertants/plate a 4-Aminobiphenyl 4-Aminobiphenyl--HC1 Benzidine Benzidine--2HC1 3,3'-Dimethylbenzidine 3,3'-Dimethylbenzidine--2HC1 3,3~-Dimethoxybenzidine 3,3~-Dimethoxybenzidine--2HCl 3.3r-Dichlorobenzidine 3,3'-Dichlorobenzidine--2HC1
613 1139 455 635 198 356 172 201 14 16 280 360 0 0 12 11 2071 2012 1612 1872
50 100 50 100 50 100 50 100 50 100 50 100 50 100 50 100 50 100 50 100
Av. Spontaneous Mutation Frequency
(320) b (622) (244) (383)
36 Range
(26--42)
1212 1456 1152 1455 57 93 114 91 3 10 64 92 0 0 7 0 62 66 66 70 137 (122--161)
a Average spontaneous mutation frequency has been subtracted from the entries. b Revertants without mammalian enzyme fractions. All of the compounds were tested, but only the positive results were reported.
benzidine with 4-aminobiphenyl. In this table, the average spontaneous mutation frequencies have been subtracted from the reported values. Where comparable data are available, the mutation frequencies reported here were slightly lower than have been reported in some other studies [7,11]. These lower values are probably due to the fact that the uninduced mouse S-9 preparations have a lower enzymatic activity level than the induced rat S-9 fractions used in other studies. The control compounds, the 4-aminobiphenyl analogs, caused both base pair substitution and frameshift mutations as indicated b y their reactivity with both tester strains. The 3,3'-dichlorobenzidine c o m p o u n d s were moderately mutagenic w i t h o u t the mammalian enzyme preparation; however, the muta-
24
genicity was increased markedly when the S-9 fraction was added to the reaction mixtures. Based on the number of revertant colonies as a measure of mutagenicity, the 3,3'-dichlorobenzidine compounds were the most active mutagens,and the 3,3'-dimethoxybenzidine analogs were the least mutagenic. All of the compounds have been reported to be carcinogenic in experimental animals, b u t they have n o t been studied extensively in mice [6,8]. To improve the solubility and stability of the c o m p o u n d s for the long-term, low dose animal feeding experiments, the compounds were dissolved in aqueous HC1 (0.01 N, pH 2). When the mutagenicities of these salts were compared with those of the parent compounds, the hydrochloric acid tended to decrease slightly the revertant colony counts. Only in the case of the 3,3'-dimelthylbenzidine did the mutagenicity increase when the hydrochloric acid was added to the compound. Animal feeding experiments are in progress or are planned to evaluate the carcinogenic potential of these hydrochloride salts of benzidine in mice. Preliminary results with benzidine and 4-aminobiphenyl suggest that there is a dose response relationship for liver neoplasias in all groups except the controls. When additional histopathological data become available, attempts will be made to correlate the in vitro results with the toxicity and carcinogenicity of all the compounds. ACKNOWLEDGEMENTS
We acknowledge the expert technical assistance of J.G. Shaddo~k, P.R. Barren and D.M. Nestorick. REFERENCES 1 Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. (1973) Carcinogens are mutagerm: A simple test system combining liver homogenates for activations and bacteria for detection. Proc. Natl. Acad. Sci. USA, 70, 2281--2285. 2 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31,346--364. 3 Barsch, H. (1976) Predictive value of mutagenicity tests in chemical carcinogenesis. Mutat. Res., 3 8 , 1 7 7 - - 1 9 0 . 4 Bowman, M.C., King, J.R. and Holder, C.L. (1976) Benzidine and congeners: Analytical chemical properties and trace analysis in five substrates. Intern. J. Environ. Anal. Chem., 4, 205--223. 5 Bowman, M.C. and Rushing, L.G. (1977) 3,3'-Dichlorobenzidine: Trace analysis in animal chow, wastewater and human urine b y three gas chromatographic procedures. Arch. Environ. Contain. Toxicol. In press. 6 Clayson, D.B. (1962) Chemical carcinogenesis. Churchill, London. 7 Garner, R.C., Walpole, A.L. and Rose, F.L. (1975) Testing of some benzidine analogues for microsomal activation to bacterial mutagens. Cancer Lett., 1, 39--42. 8 Haley, T.J. (1975) Benzidinerevisited: A r e v i e w o f the literature and problems associated with the use of benzidine and its congeners. Clin. Toxicol., 8, 13--42.
25 9 Holder, C.L., King, J.R. and Bowman, M.C. (1976) 4-Aminobiphenyl, 2-naphthylamine and analogs. Analytical properties and trace analysis in five substrates. J. Toxicol Environ. Health, 2,111--129. 10 McCann, J., Spingarn, N.E., Kobori, J. and Ames, B.N. (1975) The detection of carcino gens as mutagens: Bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Sci. ~SA, 72,979--983. 11 McCann, J., Choi, E., Yamaski, E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test. Assay of 300 chemicals. Proc. Natl. Acad. Sci. USA, 73, 5135--5139. 12 Sivik, A. (1976) The Ames assay. Science, 193,272--274.