Paller M S, Eaton J W
Department of Medicine, University of Minnesota, Minneapolis 55409-0392, USA.
Free Radic Biol Med. 1995 May;18(5):883-90. doi: 10.1016/0891-5849(94)00222-6.
Oxygen free radical scavengers protect against ischemia/reperfusion injury of the kidney in vivo and against hypoxia/reoxygenation (H/R) injury of renal cells in several in vitro systems. In an attempt to maximize renal protection we tested several antioxidants in combination; the individual components had previously reduced reoxygenation injury of hypoxic renal epithelial cells. Both glutathione (GSH; 1 mM) and Cu,Zn-SOD provided significant protection against posthypoxic injury. Surprisingly, the combination of Cu,Zn-SOD plus GSH eliminated protection entirely and was highly toxic to normoxic cells. The toxicity of Cu,Zn-SOD+GSH was not prevented by the iron chelator deferoxamine and was only slightly reduced by the hydroxyl scavenger DMTU. Catalase reversed the toxicity of Cu,Zn-SOD+GSH and provided net protection. Direct measurement of intracellular peroxides using 2,7-dichlorofluorescein quantitated by laser cytometry also revealed enhanced generation of peroxides by cells during H/R when Cu,Zn-SOD+GSH was present. GSSG was less toxic than GSH when combined with Cu,Zn-SOD. Importantly, the combination of Mn-SOD+GSH provided superior protection to either agent alone. In the presence of added GSH, heated or autoclaved Cu,Zn-SOD was still toxic, whereas SOD free of chelatable Cu++ was benign. In the presence of GSH, Cu++ derived from SOD may promote the formation of toxic thionyl radicals, metal-centered radicals, and/or H2O2, thereby causing cell injury. Great care should be used in designing and interpreting studies employing combinations of antioxidants.
氧自由基清除剂在体内可保护肾脏免受缺血/再灌注损伤，在多种体外系统中也可保护肾细胞免受缺氧/复氧（H/R）损伤。为了实现最大程度的肾脏保护，我们测试了几种抗氧化剂的组合；之前单个成分已能减轻缺氧肾上皮细胞的复氧损伤。谷胱甘肽（GSH；1 mM）和铜锌超氧化物歧化酶（Cu,Zn-SOD）均可显著保护细胞免受缺氧后损伤。令人惊讶的是，Cu,Zn-SOD与GSH的组合完全消除了保护作用，且对常氧细胞具有高毒性。铁螯合剂去铁胺不能阻止Cu,Zn-SOD+GSH的毒性，羟基清除剂二甲基硫脲（DMTU）只能略微降低其毒性。过氧化氢酶可逆转Cu,Zn-SOD+GSH的毒性并提供净保护作用。使用激光细胞术对2,7-二氯荧光素定量直接测量细胞内过氧化物，结果也显示当存在Cu,Zn-SOD+GSH时，细胞在H/R过程中过氧化物生成增加。与Cu,Zn-SOD联合使用时，氧化型谷胱甘肽（GSSG）的毒性低于GSH。重要的是，锰超氧化物歧化酶（Mn-SOD）与GSH的组合比单独使用任何一种试剂都能提供更好的保护。在添加GSH的情况下，加热或高压灭菌的Cu,Zn-SOD仍具有毒性，而不含可螯合铜离子（Cu++）的超氧化物歧化酶则无毒性。在存在GSH的情况下，超氧化物歧化酶释放的Cu++可能促进有毒的亚硫酰基自由基、金属中心自由基和/或过氧化氢的形成，从而导致细胞损伤。在设计和解释使用抗氧化剂组合的研究时应格外小心。
