Acidic horseradish peroxidase activity abolishes genotoxicity of common dyes

The aim of this study was to investigate the impact of dyes on DNA before and after enzymatic decolorization by acidic horseradish peroxidase (HRP-A). The comet assay is easy and feasible method widely used to measure DNA damage and repair. The medium-throughput comet assay was employed for assessment of genotoxic effects of 8 dyes in BEAS-2B cells. We have incorporated a digestion with bacterial endonuclease (formamidopyrimidine DNA glycosylase, FPG) to detect oxidized bases in the case of single and double azo dyes, Orange II (OR2) and Amido Black 10B (AB), respectively. This allowed detection 8-oxo-7,8-dihydroguanine, one of most abundant oxidized bases in nuclear DNA. In the case of AB there was no indication of DNA damage, either strand brakes or FPG-sensitive sites before and after decolorization. The OR2 induced DNA damage (in terms of percentage of DNA in comet tails). Also, the frequency of FPG-sensitive sites increased with OR2 concentration. After decolorization no DNA damaging effects was seen at all. The interaction studies of OR2 and AB, before and after decolorization, with calf thymus DNA has been investigated by absorption and fluorescence spectroscopy. The results provide support for the idea that in some cases enzymatic decolorization contributes to lower genotoxicity potential.