Horseradish peroxidase mediates DNA and deoxyguanosine 3'-monophosphate adduct formation in the presence of ochratoxin A

Abstract.

Ochratoxin A (OTA) gives rise to DNA and deoxyguanosine-3'-monophosphate (dGMP) adducts in vitro using mice kidney microsomes in the presence of arachidonic acid. This result points to the involvement of prostaglandin H synthases, which are present at high levels in the kidney, urinary bladder and seminal vesicles, and/or of lipoxygenases in the metabolic activation of OTA to genotoxic compounds. These enzymes have peroxidase activities. Incubation of OTA with DNA in the presence of horseradish peroxidase (HRP) and cumene hydroperoxide at pH 7.4 led to the formation of one major and three minor adducts with a total adduct level of 42 per 10⁹ nucleotides. Incubation with dGMP gave a total adduct level of 159 per 10⁹ nucleotides. In the presence of H₂O₂ instead of cumene hydroperoxide, a lower level of adducts was obtained, both with DNA and dGMP. The concentrations of HRP and co-substrate used in this paper were higher than those used by other authors who obtained negative results when they sought DNA adducts of OTA in the presence of HRP and H₂O₂. The main adduct we obtained with DNA incubated with HRP and OTA had the same chromatographic behaviour as that obtained when DNA or dGMP were incubated with OTA, arachidonic acid and mice kidney microsomes. However, the main adduct obtained with dGMP incubated with HRP and OTA behaved differently. These results show that OTA can be metabolized by a peroxidase to metabolically activated species that bind covalently to DNA and dGMP; however, the main adduct obtained in vitro with HRP and dGMP cannot serve as a model for one of the adducts formed by OTA with DNA because it behaves differently in two chromatographic systems.