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The interpretation of bacterial mutation tests with Azo dyes
Mutation Research, 91 (1981) 153-154
153
~c) Elsevier/North-Holland Biomedical Press
LETTER TO THE EDITOR
T H E I N T E R P R E T A T I O N OF B A C T E R I A L M U T A T I O N TESTS W I T H AZO DYES
Dear Sir, In a recent paper published in this journal (S. Venturini and M. Tamaro, Mutation Res., 68 (1979) 307-312) the results of screening several azo textile dyes in the Ames assay were presented. 3 of the colours which induced frame-shift mutations did not require activation with $9 mix and these authors concluded that mutagenicity was the property of the unreduced dyes. In 2 cases activity was tentatively attributed to the presence of amino groups. One monoazo colour, Red 2BF, with a free amino group was non-mutagenic. We have found at least one monoazo dye possessing an amino substituent to be direct-acting, although, in this case transitions were induced. With Red 10B (C.I. 17200) this activity was abolished by chemical reduction and we believe that the intact molecule is directly active since it is microsomally deactivated and is resistant to tester-strain mediated reduction (R.B. Haveland-Smith and R.D. Combes, Fd. Cosmet. Toxicol., 18 (1980) in press). The results with the textile dyes mentioned are interesting. It would seem that no significant reduction was occurring since 3 of the inactive dyes (C.I. Acid Reds 111, 85 and 114) would be expected to yield benzidine or 2,2'-dimethylbenzidine (closely related to o-tolidine: 3,3'-dimethylbenzidine), which are known or likely to be indirect frame-shift mutagens (E. Lavoie et al., Mutation Res., 67 (1979) 123; C.P. Hartman et al., Mutation Res., 58 (1978) 125). What is more surprising is that these dyes could not be activated by riboflavin-enriched $9 mix. Other benzidine-based disazo dyes such as Trypan Blue, Benzopurpurine 4B, Congo Red and Chlorazol Violet N can be activated in this way, or by chemical or microbially-mediated reduction (C.P. Hartman et al., ibid.; T. Matsushima et al., Mutation Res., 54 (1978) 220). Evans Blue is the only other exception in the literature known to me. This dye remained inactive following incubation with dithionite and $9 (J.P. Brown et al., Mutation Res., 56 (1978) 249). How do we interpret in vitro mutagenicity assays revealing negative data for chemicals which are structurally similar to compounds demonstrated to be metabolised in vivo to known genotoxicants? In the present context we must consider the likelihood that azo dyes will be reduced in the particular genetic system being utilized; since in vivo reduction of benzidine disazo dyes to benzidine has been demonstrated (E. Rinde and W. Troll, J. Natl. Cancer Inst., 55 (1975) 181). This is especially relevant if urinary benzidine in dyestuff workers is responsible for occupational bladder cancers (see J.L. Radomski, Ann. Rev. Pharmacol. Toxicol., 19 (1979) 129). Studies with monoazo dyes suggest that low electron densities in the
154
azo bond region favour reduction (R. Walker and A.J. Ryan, Xenobiotica, 1 (1971) 483). Such a situation may be brought about by hydrogen-bonding between an azo nitrogen-atom and an adjacent naphthol group. With disazo dyes overall reduction rates may be slower due to the extra azo bond. However, if the same criterion applies this hydrogen bonding might occur in all 3 of the inactive textile dyes. Furthermore, this bonding is unlikely with Trypan Blue due to preferential hydrogen bonding of the hydroxyl groups with the adjacent more basic naphthylamino groups. The other disazo dyes mentioned lack such hydroxyl groups, having in their place amino substituents proximal to the azo bond with less probability of hydrogen bonding. Thus electron densities in the azo region will be higher. These observations suggest that the textile disazo dyes should have been more easily reduced than they were, especially with added riboflavin. It is feasible that the $9 mix prepared by Venturini and Tamaro possessed low or non-existent azo-reductase activity or was unable to activate the amines liberated. It is recommended that the susceptibility of azo dyes to reduction during testing in bacterial mutagenicity assays is closely investigated. This will necessitate a knowledge of the ability of the tester strains to metabolise the dyes. Also colours such as chrysoidine, a mutagenic dye one of whose reduction products is activated by standard $9 mix (R.C. Garner and C.A. Nutman, Mutation Res., 44 (1977) 9) and benzopurpurine which requires addition of riboflavin to $9 mix should be used as positive controls to check the activity of the various metabolising systems employed. When structural features suggest genotoxicity of breakdown products and such dyes are inactive in these assays, chemical and specific gut microbial activation should also be utilized. If azo colours, such as the inactive textile dyes referred to, remain inactive after such pre-treatments involving efficient reduction, then this would significantly enhance the value of the negative results previously obtained. Since certain azo colours may be detoxified by reduction, routine supplementation of $9 mix with extra riboflavin is not recommended since it may lead to an incomplete assessment of genotoxicity. I am not suggesting that structure alone can be used to predict genotoxicity. However, the results of screening with Ames and other in vitro assays should be discussed with reference to molecular structure and information concerning the possible metabolites formed from the agents in question or from similar compounds. It should be remembered that many chemicals subjected to mutagenicity testing may never be monitored for animal carcinogenicity unless they are Ames-positive and are of some benefit commercially or otherwise. If the Ames assay is to be used as an objective pre-screen for potential genotoxic agents the interpretation of positive and negative data must receive equal consideration using knowledge concerning in vivo mammalian metabolism whenever possible. Yours sincerely, R.D. COMBES
Department of Biological Sciences, Portsmouth Polytechnic, King Henry I Street, Portsmouth POI 2DY (Great Britain) (Accepted 15 October 1980)
153
~c) Elsevier/North-Holland Biomedical Press
LETTER TO THE EDITOR
T H E I N T E R P R E T A T I O N OF B A C T E R I A L M U T A T I O N TESTS W I T H AZO DYES
Dear Sir, In a recent paper published in this journal (S. Venturini and M. Tamaro, Mutation Res., 68 (1979) 307-312) the results of screening several azo textile dyes in the Ames assay were presented. 3 of the colours which induced frame-shift mutations did not require activation with $9 mix and these authors concluded that mutagenicity was the property of the unreduced dyes. In 2 cases activity was tentatively attributed to the presence of amino groups. One monoazo colour, Red 2BF, with a free amino group was non-mutagenic. We have found at least one monoazo dye possessing an amino substituent to be direct-acting, although, in this case transitions were induced. With Red 10B (C.I. 17200) this activity was abolished by chemical reduction and we believe that the intact molecule is directly active since it is microsomally deactivated and is resistant to tester-strain mediated reduction (R.B. Haveland-Smith and R.D. Combes, Fd. Cosmet. Toxicol., 18 (1980) in press). The results with the textile dyes mentioned are interesting. It would seem that no significant reduction was occurring since 3 of the inactive dyes (C.I. Acid Reds 111, 85 and 114) would be expected to yield benzidine or 2,2'-dimethylbenzidine (closely related to o-tolidine: 3,3'-dimethylbenzidine), which are known or likely to be indirect frame-shift mutagens (E. Lavoie et al., Mutation Res., 67 (1979) 123; C.P. Hartman et al., Mutation Res., 58 (1978) 125). What is more surprising is that these dyes could not be activated by riboflavin-enriched $9 mix. Other benzidine-based disazo dyes such as Trypan Blue, Benzopurpurine 4B, Congo Red and Chlorazol Violet N can be activated in this way, or by chemical or microbially-mediated reduction (C.P. Hartman et al., ibid.; T. Matsushima et al., Mutation Res., 54 (1978) 220). Evans Blue is the only other exception in the literature known to me. This dye remained inactive following incubation with dithionite and $9 (J.P. Brown et al., Mutation Res., 56 (1978) 249). How do we interpret in vitro mutagenicity assays revealing negative data for chemicals which are structurally similar to compounds demonstrated to be metabolised in vivo to known genotoxicants? In the present context we must consider the likelihood that azo dyes will be reduced in the particular genetic system being utilized; since in vivo reduction of benzidine disazo dyes to benzidine has been demonstrated (E. Rinde and W. Troll, J. Natl. Cancer Inst., 55 (1975) 181). This is especially relevant if urinary benzidine in dyestuff workers is responsible for occupational bladder cancers (see J.L. Radomski, Ann. Rev. Pharmacol. Toxicol., 19 (1979) 129). Studies with monoazo dyes suggest that low electron densities in the
154
azo bond region favour reduction (R. Walker and A.J. Ryan, Xenobiotica, 1 (1971) 483). Such a situation may be brought about by hydrogen-bonding between an azo nitrogen-atom and an adjacent naphthol group. With disazo dyes overall reduction rates may be slower due to the extra azo bond. However, if the same criterion applies this hydrogen bonding might occur in all 3 of the inactive textile dyes. Furthermore, this bonding is unlikely with Trypan Blue due to preferential hydrogen bonding of the hydroxyl groups with the adjacent more basic naphthylamino groups. The other disazo dyes mentioned lack such hydroxyl groups, having in their place amino substituents proximal to the azo bond with less probability of hydrogen bonding. Thus electron densities in the azo region will be higher. These observations suggest that the textile disazo dyes should have been more easily reduced than they were, especially with added riboflavin. It is feasible that the $9 mix prepared by Venturini and Tamaro possessed low or non-existent azo-reductase activity or was unable to activate the amines liberated. It is recommended that the susceptibility of azo dyes to reduction during testing in bacterial mutagenicity assays is closely investigated. This will necessitate a knowledge of the ability of the tester strains to metabolise the dyes. Also colours such as chrysoidine, a mutagenic dye one of whose reduction products is activated by standard $9 mix (R.C. Garner and C.A. Nutman, Mutation Res., 44 (1977) 9) and benzopurpurine which requires addition of riboflavin to $9 mix should be used as positive controls to check the activity of the various metabolising systems employed. When structural features suggest genotoxicity of breakdown products and such dyes are inactive in these assays, chemical and specific gut microbial activation should also be utilized. If azo colours, such as the inactive textile dyes referred to, remain inactive after such pre-treatments involving efficient reduction, then this would significantly enhance the value of the negative results previously obtained. Since certain azo colours may be detoxified by reduction, routine supplementation of $9 mix with extra riboflavin is not recommended since it may lead to an incomplete assessment of genotoxicity. I am not suggesting that structure alone can be used to predict genotoxicity. However, the results of screening with Ames and other in vitro assays should be discussed with reference to molecular structure and information concerning the possible metabolites formed from the agents in question or from similar compounds. It should be remembered that many chemicals subjected to mutagenicity testing may never be monitored for animal carcinogenicity unless they are Ames-positive and are of some benefit commercially or otherwise. If the Ames assay is to be used as an objective pre-screen for potential genotoxic agents the interpretation of positive and negative data must receive equal consideration using knowledge concerning in vivo mammalian metabolism whenever possible. Yours sincerely, R.D. COMBES
Department of Biological Sciences, Portsmouth Polytechnic, King Henry I Street, Portsmouth POI 2DY (Great Britain) (Accepted 15 October 1980)