Abstract
A recently developed sensitive fluorimetric assay has been used to examine whether free hydroxyl radicals (HO') 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 H 2O 2, 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.
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