Hazards of antioxidant combinations containing superoxide dismutase

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-dichlorofluorescin 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.