Ueda J, Takai M, Shimazu Y, Ozawa T
National Institute of Radiological Sciences, Chiba-shi, Japan.
Arch Biochem Biophys. 1998 Sep 15;357(2):231-9. doi: 10.1006/abbi.1998.0811.
The generation of hydroxyl radicals (.OH) from the reaction of Cu(II) complexes with biological reductants such as ascorbic acid, glutathione, acetylcysteine, and hydroquinone was confirmed by spin-trapping experiments using electron spin resonance (ESR). The following Cu(II) complexes were used: Cu(II)-(CyHH)2 (CyHH, cyclo(L-histidyl-L-histidyl)), Cu(II)(OP)2 (OP, o-phenanthroline), Cu(II)(HGG) (HGG, L-histidyl-glycylglycine), and Cu(II)(en)2 (en, ethylenediamine). The methyl radical adduct of alpha-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN-CH3) was obtained from the reaction of ascorbic acid with all Cu(II) complexes used here in the presence of a spin trap, POBN, and dimethyl sulfoxide, indicating the generation of .OH. Glutathione, N-acetylcysteine, and hydroquinone reacted with both Cu(II)(CyHH)2 and Cu(II)(OP)2 to generate POBN-CH3, while these reductants did not react with either Cu(II)(HGG) or Cu(II)(en)2. Interestingly, the formation of POBN-CH3 in the reaction of Cu(II)-(CyHH)2 with glutathione or N-acetylcysteine was found only at a Cu(II)(CyHH)2/glutathione or Cu(II)(CyHH)2/N-acetylcysteine ratio of 1. The DNA strand scission caused by reaction mixtures of Cu(II) complexes with reductants was investigated under the same conditions as the ESR spin-trapping experiments. Addition of ascorbic acid to mixtures of these four Cu(II) complexes and DNA resulted in DNA strand breakage. Hydroquinone plus Cu(II)(CyHH)2 also caused DNA strand scission. In addition, DNA strand breakage was observed with the reaction of Cu(II)(OP)2 with glutathione, N-acetylcysteine, and hydroquinone. In contrast, reaction mixtures of glutathione, N-acetylcysteine, or hydroquinone with Cu(II)-(HGG) or Cu(II)(en)2 did not cause DNA strand scission within the concentration range used. The results obtained here suggest that there is a good correlation between POBN-CH3 formation and DNA strand scission. Thus, DNA strand scission may be caused by .OH generated from the reaction of some Cu(II) complexes with biological reductants under aerobic conditions. Since ascorbic acid, glutathione, and N-acetylcysteine are present in living cells, some Cu(II) complexes may be capable of initiating DNA damage in the presence of these reductants.
通过使用电子自旋共振（ESR）的自旋捕获实验，证实了铜（II）配合物与生物还原剂（如抗坏血酸、谷胱甘肽、乙酰半胱氨酸和对苯二酚）反应生成羟基自由基（·OH）。使用了以下铜（II）配合物：铜（II）-（CyHH）2（CyHH，环（L-组氨酰-L-组氨酰））、铜（II）（OP）2（OP，邻菲罗啉）、铜（II）（HGG）（HGG，L-组氨酰-甘氨酰甘氨酸）和铜（II）（en）2（en，乙二胺）。在自旋捕获剂POBN和二甲基亚砜存在的情况下，抗坏血酸与这里使用的所有铜（II）配合物反应，得到α-（吡啶-4-N-氧化物）-N-叔丁基硝酮（POBN-CH3）的甲基自由基加合物，表明生成了·OH。谷胱甘肽、N-乙酰半胱氨酸和对苯二酚与铜（II）（CyHH）2和铜（II）（OP）2都反应生成POBN-CH3，而这些还原剂与铜（II）（HGG）或铜（II）（en）2都不反应。有趣的是，仅在铜（II）-（CyHH）2/谷胱甘肽或铜（II）-（CyHH）2/N-乙酰半胱氨酸的比例为1时，才发现铜（II）-（CyHH）2与谷胱甘肽或N-乙酰半胱氨酸反应生成POBN-CH3。在与ESR自旋捕获实验相同的条件下，研究了铜（II）配合物与还原剂的反应混合物引起的DNA链断裂。向这四种铜（II）配合物与DNA的混合物中加入抗坏血酸会导致DNA链断裂。对苯二酚加铜（II）（CyHH）2也会导致DNA链断裂。此外，观察到铜（II）（OP）2与谷胱甘肽、N-乙酰半胱氨酸和对苯二酚反应会导致DNA链断裂。相比之下，在所用浓度范围内，谷胱甘肽、N-乙酰半胱氨酸或对苯二酚与铜（II）-（HGG）或铜（II）（en）2的反应混合物不会导致DNA链断裂。这里获得的结果表明，POBN-CH3的形成与DNA链断裂之间存在良好的相关性。因此，DNA链断裂可能是由一些铜（II）配合物在有氧条件下与生物还原剂反应生成的·OH引起的。由于抗坏血酸、谷胱甘肽和N-乙酰半胱氨酸存在于活细胞中，一些铜（II）配合物在这些还原剂存在的情况下可能能够引发DNA损伤。
