ABSTRACT
We have evaluated the mutagenic activity of a series of diazo compounds derived from benzidine and its congeners o-tolidine, o-dianisidine and 3,3'-dichlorobenzidine as well as several monoazo compounds. The test system used was a modification of the standard Ames Salmonella assay in which FMN, hamster liver S9 and a preincubation step are used to facilitate azo reduction and detection of the resulting mutagenic aromatic amines. All of the benzidine and o-tolidine dyes tested were clearly mutagenic. The o-dianisidine dyes except for Direct Blue 218 were also mutagenic. Direct Blue 218 is a copper complex of the mutagenic o-dianisidine dye Direct Blue 15. Pigment Yellow 12, which is derived from 3,3'-dichlorobenzidine, could not be detected as mutagenic, presumably because of its lack of solubility in the test reaction mixture. Of the monoazo dyes tested, methyl orange was clearly mutagenic, while C.I. Acid Red 26 and Acid Dye (C.I. 16155; often referred to as Ponceau 3R) had marginal to weak mutagenic activity. Several commercial dye samples had greater mutagenic activity with the modified test protocol than did equimolar quantities of their mutagenic aromatic amine reduction products. Investigation of this phenomenon for Direct Black 38 and trypan blue showed that it was due to the presence of mutagenic impurities in these samples. The modified method used appears to be suitable for testing the mutagenicity of azo dyes, and it may also be useful for monitoring the presence of mutagenic or potentially carcinogenic impurities in otherwise nonmutagenic azo dyes.
Subject(s)
Azo Compounds/pharmacology , Benzidines/pharmacology , Coloring Agents/toxicity , Mutation/drug effects , 3,3'-Dichlorobenzidine/pharmacology , Dianisidine/pharmacology , Mutagenicity Tests , Salmonella typhimurium/drug effects , Structure-Activity RelationshipABSTRACT
A protocol for assessing the mutagenic activity of azo dyes derived from mutagenic or potentially mutagenic aromatic amines was evaluated, using 4 model compounds. This protocol is based upon one developed in Sugimura's laboratory with modifications, including the use of flavin mononucleotide (FMN) rather than riboflavin to reduce the azo compounds to free amines, and hamster liver S9 rather then rat liver S9 for metabolic activation. The protocol developed differs from the standard Ames Salmonella plate incorporation assay in 5 ways: (1) uninduced hamster liver S9 rather than Aroclor 1254-induced rat liver S9 is used; (2) 150 microliters of S9 is used rather than the maximum of 50 microliter of S9 used in the standard assay; (3) FMN is added to the cofactor mix; (4) the cofactor mix is modified to include exogenous glucose 6-phosphate dehydrogenase, NADH, and 4 times the standard amount of glucose 6-phosphate; and (5) a 30-min "pre-incubation" step is used before addition of top agar. We found that each of these 5 changes is necessary for optimal mutagenic activity of azo dyes derived from the mutagenic aromatic amines benzidine, o-tolidine or o-dianisidine. The use of hamster liver S9 rather than rat liver S9 was also required for optimal mutagenic activity of benzidine itself. Rat liver S9 inhibited the ability of hamster S9 to activate benzidine to a mutagen. The presence in rat liver S9 of an inhibitor of the metabolic activation of benzidine may account for the failure of benzidine and a benzidine dye (Congo red) to be strongly mutagenic when tested with this type of S9.
Subject(s)
Azo Compounds/pharmacology , Coloring Agents/pharmacology , Flavin Mononucleotide , Mutagenicity Tests/methods , Dose-Response Relationship, Drug , Microsomes, Liver/metabolism , Salmonella typhimurium/geneticsABSTRACT
Dimethylnitrosamine (DMN) was mutagenic in the Salmonella plate incorporation assay (Ames test) at a level of 10 mumol/plate (3.7 mM) in the presence of hamster liver S-9. Mutagenicity of DMN at this level was not observed when the S-9 was derived from mouse or rat liver, although the mouse liver and hamster liver S-9 had similar DMN demethylase activities. Both mouse and rat liver S-9 inhibited the mutagenicity of DMN mediated by hamster liver S-9; the inhibitory factor was contained in the microsomal fraction. Mouse or rat liver microsomes did not inhibit the DMN demethylase activity of hamster liver S-9. The microsomal inhibitor from rat or mouse liver was stable at 60 but was inactivated at 70 degrees. DMN demethylase from both rat and mouse liver was inactivated at 60 degrees. Although the DMN demethylase activity of hamster liver S-9 was contained in the microsomal fraction, DMN mutagenesis under conditions of the assay required the presence of both microsomal and cytosolic (S-105) fractions; the cytosols from hamsters, mice, and rats were all effective. The cytosolic factor required for DMN mutagenesis was sensitive to trypsin and was not dialyzable. The presence of an inhibitor of DMN activation in rat and mouse microsomes may account for, or contribute to, the failure of liver S-9 preparations from these species to activate DMN to a mutagen under standard conditions of the Ames test. The requirement for the cytosolic fraction may indicate that DMN demethylase is not sufficient for the activation of DMN to a mutagen under the conditions used in these studies.
Subject(s)
Cytosol/metabolism , Dimethylnitrosamine/pharmacology , Microsomes, Liver/metabolism , Mutagenicity Tests/methods , Salmonella typhimurium/drug effects , Animals , Aroclors , Biotransformation , Cell Fractionation , Cricetinae , Cytosol/drug effects , Dimethylnitrosamine/metabolism , Methods , Mice , Microsomes, Liver/drug effects , Mutagens , Phenobarbital/pharmacology , Rats , Salmonella typhimurium/geneticsABSTRACT
An ingredient recently introduced in hair dyes, 4-ethoxy-m-phenylenediamine, is mutagenic in histidine-requiring strains of Salmonella typhimurium. Its mutagenic activity is similar to that of the hair dye ingredient is apparently replaced, 4-methoxy-m-phenylenediamine.
