Measurement of hydroxyl radicals catalyzed in the immediate vicinity of DNA by metal-bleomycin complexes

A recently developed sensitive fluorimetric assay has been used to examine whether free hydroxyl radicals (HOʹ) are generated in the immediate vicinity of DNA by Fe(II)-bleomycin. When aqueous solutions of SECCA (the succinimidyl ester of coumarin-3-carboxylic acid) are irradiated with gamma rays or incubated with Fe(II)-bleomycin or Fe(II) or Fe(II)-EDTA in the presence of ascorbate and H2O2, 7-hydroxy-SECCA, a fluorescent product of the interaction of HO· with SECCA, is generated. Studies with catalase and several HO· scavengers indicate that the fluorescence induction is mediated by HO·. On the contrary, Cu(II)-bleomycin complexes under similar conditions fail to induce 7-hydroxy-SECCA fluorescence. When SECCA is conjugated to DNA via SECCA-polylysine-DNA complexes and incubated in the same iron-containing systems, the relative ability of the scavengers to reduce the fluorescence again demonstrates the generation of 7-hydroxy-SECCA by HO·. However, while the fluorescence is practically eliminated by high concentrations of DMSO (100 μmol dm−3) in the systems with Fe(II) or Fe(II)-EDTA, it is not possible to reduce it similarly in the case of Fe(II)-bleomycin. These data demonstrate the generation of HO· by Fe(II)-bleomycin in the immediate vicinity of DNA. Because the experiments simulate the lifetime of HO· expected in cells, these data suggest that, if such DNA-associated HO· radicals are also produced in vivo by bleomycin, these would not be scavengable by intracellular scavengers and they could interact with chromatin.