Yamamoto K, Kawanishi S
Department of Public Health, Faculty of Medicine, Kyoto University, Japan.
J Biol Chem. 1989 Sep 15;264(26):15435-40.
Site-specific DNA damage by Cu(II) plus H2O2 was investigated by a DNA-sequencing technique. Cu(II) plus H2O2 induced strong DNA cleavage even without piperidine treatment. Piperidine-labile sites were induced frequently at thymine and guanine residues and rarely at adenine residue. A Cu(I)-specific chelating agent, bathocuproine, inhibited the DNA damage. Neither ethanol nor mannitol inhibited it. Of alcohols, tertbutyl alcohol, having relatively low reactivity to hydroxyl free radical, inhibited the DNA damage most strongly. Sodium azide and 1,4-diazobicyclo[2.2.2]octane completely inhibited cleavages at residues of the bases other than guanine. Tris inhibited the DNA damage. The enhancing effect of D2O on DNA damage was not observed. ESR studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) showed that the hydroxyl radical adduct of DMPO was formed during the reaction of Cu(II) with H2O2, and that the addition of sodium formate produced the CO2- radical adduct of DMPO more efficiently than expected. ESR studies showed that the nitroxide radical was formed from 2,2,6,6-tetramethyl-4-piperidone in the presence of Cu(II) plus H2O2, indicating the formation of singlet oxygen or its equivalent. The effects of scavengers on DNA damage have considerable correlation with the effects of scavengers on the nitroxide radical production and DMPO.OH formation. The results suggest that the main active species causing DNA damage are more likely copper-peroxide complexes, with similar reactivity to singlet oxygen and/or hydroxyl radical rather than hydroxyl free radical.
采用DNA测序技术研究了Cu(II)加H2O2造成的位点特异性DNA损伤。即使不经过哌啶处理，Cu(II)加H2O2也能诱导强烈的DNA裂解。哌啶敏感位点在胸腺嘧啶和鸟嘌呤残基处频繁诱导产生，而在腺嘌呤残基处很少出现。一种Cu(I)特异性螯合剂，bathocuproine，可抑制DNA损伤。乙醇和甘露醇均不能抑制该损伤。在醇类中，对羟基自由基反应性相对较低的叔丁醇对DNA损伤的抑制作用最强。叠氮化钠和1,4-二氮杂双环[2.2.2]辛烷完全抑制了除鸟嘌呤以外的碱基残基处的裂解。Tris抑制了DNA损伤。未观察到D2O对DNA损伤的增强作用。使用5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）的电子自旋共振（ESR）研究表明，在Cu(II)与H2O2反应过程中形成了DMPO的羟基自由基加合物，并且添加甲酸钠比预期更有效地产生了DMPO的CO2-自由基加合物。ESR研究表明，在Cu(II)加H2O2存在的情况下，2,2,6,6-四甲基-4-哌啶酮形成了氮氧化物自由基，表明形成了单线态氧或其等效物。清除剂对DNA损伤的影响与清除剂对氮氧化物自由基产生和DMPO·OH形成的影响具有显著相关性。结果表明，导致DNA损伤的主要活性物种更可能是过氧化铜配合物，其反应性与单线态氧和/或羟基自由基相似，而不是羟基自由基。
