HYDROXYL RADICAL GENERATION DURING MITOCHONDRIAL ELECTRON-TRANSFER AND THE FORMATION OF 8-HYDROXYDESOXYGUANOSINE IN MITOCHONDRIAL-DNA

Production of hydroxyl radicals (HO.) by substrate-supplemented beef h eart submitochondrial particles was studied by electron paramagnetic r esonance in conjunction with the spin trap 5,5'-dimethyl-1-pirroline-N -oxide (DMPO), Supplementation of submitochondrial particles with NADH or succinate in the presence of antimycin resulted in the formation h ydroxyl-, alpha-hydroxyethyl-, and methyl radical adducts, The latter two adducts were derived from HO. attack of ethanol or dimethyl sulfox ide (DMSO), respectively, the solvents used for the inhibitors of the respiratory chain. These ESR signals were slightly increased by supero xide dismutase and abolished by catalase, Further support for the prod uction of HO. during mitochondrial electron transfer was furnished by kinetic competition experiments with DMSO as the HO. scavenger, This a pproach yielded a k(SCAVENGER)/k(DMPO) value of 1.7, in agreement with a competitive spin trapping of free HO. using DMSO as a scavenger. Th e scission of H2O2 to HO. requires consideration of a Fenton chemistry , i,e., the participation of metals or redox active metal pools in mit ochondria to drive this reaction, The effect of several metal chelator s on the formation of both HO. and H2O2 was examined. Bathophenantroli ne, bathocuproine, and desferrioxamine decreased the DMPO-HO. signal a nd increased accumulation of H2O2. Conversely, EDTA or diethylenetriam inepentaacetic acid substantially increased the DMPO-HO. signal intens ity and decreased H2O2 accumulation, These different results were rati onalized in terms of the reduction potential of the redox couples invo lved, i,e., that of the ligated metal and those encompassed in the one -electron reduction of superoxide radical and of hydrogen peroxide. Th e formation of 8-hydroxydesoxyguanosine in mitochondrial DNA was exami ned under experimental conditions in which H2O2 production by isolated mitochondria was enhanced. The formation of 8-hydroxydesoxyguanosine increased with increasing rates of H2O2 formation. The biological sign ificance of H2O2 and HO. formation during mitochondrial electron trans fer is discussed in terms of oxidative damage of mitochondrial DNA and the implications for mitochondrial functions and aging. (C) 1995 Acad emic Press, Inc.