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.

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