Mechanism of formation and 32P-postlabeling of DNA adducts derived from peroxidative activation of carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene (Sudan I)

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Abstract
Horseradish peroxidase in the presence of hydrogen peroxide mediates the activation of carcinogenic 1-phenyl-azo-2-hydroxynaphthalene (Sudan I) to DNA-bound products in vitro. The peroxidase activating system is > 10 times more effective with respect to DNA modification by Sudan I than the microsomal enzymes containing cytochrome P450. The DNA-binding reaction of the Sudan I metabolite(s) formed by the peroxidase system is dependent on Sudan I and H2O2 concentration and pH. Reactive intermediate(s) or product(s) of the Sudan I oxidation by peroxidase with a short half-life are responsible for the DNA modification. DNA modified by peroxidase-activated Sudan I becomes colored and has an absorption maximum at ˜ 480 nm. The modification of DNA by Sudan I metabolites(s) formed by the peroxidase system is inhibited by some compounds of physiological importance (ascorbate, glutathione, Mg2± ions) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-Postlabeling assay of the DNA modified by Sudan I activated by the peroxidase system indicates that the covalent DNA adduct formation is the principal type of the DNA modification. Four major and several minor adducts of deoxyribonucleotide 3',5'-bisphosphate from DNA with Sudan I metabolite(s) were detected by the classical Randerath 32P-postlabelling assay as well as by the nuclease P1 version of the same method.

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