Chakrabarti S, Makrigiorgos G M, O'Brien K, Bump E, Kassis A I
Department of Radiation Oncology, Harvard Medical School, Boston, MA, USA.
Free Radic Biol Med. 1996;20(6):777-83. doi: 10.1016/0891-5849(95)02160-4.
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)-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 mumols 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.
一种最近开发的灵敏荧光测定法已被用于检测Fe(II)-博来霉素是否在DNA紧邻区域产生游离羟基自由基(HO·)。当用γ射线照射香豆素-3-羧酸琥珀酰亚胺酯(SECCA)的水溶液，或在抗坏血酸和H2O2存在的情况下，将其与Fe(II)-博来霉素或Fe(II)-乙二胺四乙酸(EDTA)一起孵育时，会产生7-羟基-SECCA，这是HO·与SECCA相互作用的荧光产物。用过氧化氢酶和几种HO·清除剂进行的研究表明，荧光诱导是由HO·介导的。相反，在类似条件下，Cu(II)-博来霉素复合物不能诱导7-羟基-SECCA荧光。当SECCA通过SECCA-聚赖氨酸-DNA复合物与DNA结合，并在相同的含铁体系中孵育时，清除剂降低荧光的相对能力再次证明了HO·产生了7-羟基-SECCA。然而，虽然在含有Fe(II)或Fe(II)-EDTA的体系中，高浓度的二甲基亚砜(DMSO，100 μmol dm-3)几乎消除了荧光，但在Fe(II)-博来霉素的情况下，却无法以类似方式降低荧光。这些数据证明了Fe(II)-博来霉素在DNA紧邻区域产生了HO·。由于实验模拟了细胞中预期的HO·寿命，这些数据表明，如果博来霉素在体内也产生这种与DNA相关的HO·自由基，那么这些自由基不能被细胞内的清除剂清除，并且它们可能与染色质相互作用。
