Measurement of copper-dependent oxidative DNA damage by HOCl and H2O2 with the ethidium-binding assay

The ETB-binding assay provides a rapid means to investigate time profiles for oxidative degradation of double-stranded DNA, by detecting the loss of ability of DNA to form a fluorescent intercalation complex with ETB. The assay was applied to demonstrate copper-dependent damage to DNA by HOCl using ascorbate as reductant. DNA degradation in this system proceeds with a rate comparable to that of reaction of HOCl with the DNA-Cu(I) complex, as monitored by the loss of DNA-Cu(I) absorption. The reaction of HOCl with DNA-Cu(I) is more than two orders of magnitude faster than the reaction of H2O2 with DNA-Cu(I). HOCl presumably reacts like H2O2 with DNA-Cu(I) to generate strongly oxidizing OH radicals immediately at the Cu(I) binding site. Quenching of the DNA/ETB fluorescence by DNA-bound copper was investigated because this may interfere with detection of copper-dependent damage to DNA with the ETB-binding assay, if not appropriate chelators are applied to remove the copper from the DNA.