Studies on the activation of azo-dyes into direct-acting genotoxic agents by Enterococcus faecalis
The ability of the intestinal bacterium Enterococcus faecalis to reduce a range of azo dyes was studied and the optimal pH and temperature for azoreductase activity were determined. The rate of dye reduction was highly variable between dyes. No obvious correlation existed between chemical structure of dye and reduction rate. Both glucose and lactate acted as electron donors for dye reduction. lodoacetate, HQNO and PCMB inhibited azo reduction whereas sodium azide did not, thus demonstrating that azoreduction involves a flavin-based electron transport system. The genotoxic and mutagenic activity of the dyes was investigated prior to and following reduction by Ent. faecalis using a variety of assays. Again there was considerable variation among the dyes tested with regard to the production of genotoxic and mutagenic agents. Although the majority of dyes showed little or no mutagenicity in the Salmonella/mutagenicity assay (Ames test), the M13 mutagenesis assay and the SOS Chromotest many were active using the Fluctuation assay. These dyes were also highly genotoxic using a differential killing assay with repair-proficient and repair-deficient E. coli strains. Although it was thought that reductive cleavage of the azo-compound generates metabolites that are able to damage DNA, activation was not always dependent on dye reduction by Ent. faecalis. While mutagenicity was not observed in the M13 mutagenesis assay the rate of bacteriophage transfection was lowered after exposure to the dyes under certain conditions, which may again be a reflection of DNA damage. The precise nature and significance of this damage in terms of toxicity and/or carcinogenicity remains to be determined. The electrochemical characteristics of the azo dyes were studied in aqueous buffer systems. Upon electrolytic reduction of the dyes a single irreversible reduction process was observed when using repeat scan cyclic voltammetry (CV). The reduction potentials of the first forward wave could be roughly correlated with the bacterial metabolism of the dyes as measured spectrophotometrically. Although a reverse wave for the azo-reduction process was absent, an associated oxidation wave was frequently observed in the return sweep of the CV due to the formation of a new redox-active species. No data has been obtained to suggest that the damaging compound produced upon reductive cleavage of the azo bond or the mechanism of genotoxicity is related to the new redox-active complex.