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PTW07 Poster Session 18 ‐ Demyelination

This study was aimed at establishing whether oxidative stress induced by acute depletion of brain glutathione (GSH) is sufficient to generate protein carbonyls (PCOs). To this end, rat brain slices were incubated separately with the GSH depletors 1,3-bis [2chloroethyl]-1-nitrosourea (BCNU) and diethyl maleate (DEM), and protein carbonylation was assessed on western blots after derivatization with dinitrophenyl hydrazine. Incubation with 1mM BCNU or 10 mM DEM for 2 h decreased GSH levels by >70%. Under these conditions the carbonylation of several proteins (40– 120 kDa) increased by 2–3 fold. The iron chelator deferoxamine, the superoxide scavenger rutin and the H2O2 quencher dimethylthiourea all prevented DEM-induced protein carbonylation, indicating that the underlying mechanism involves the iron-catalyzed generation of hydroxyl radicals from H2O2. Inhibition of catalase activity with sodium azide and aminotriazole, and glutathione peroxidase activity with mercaptosuccinic acid did not augment the amount of PCOs, suggesting that increased production of reactive oxygen species (ROS) rather than compromised antioxidant defenses is the cause for the accumulation of H2O2 after GSH depletion. PCO formation was unaffected by the xanthine oxidase inhibitor oxypurinol but it was reduced by SKF-525A and CCCP, indicating that the microsomal monoxygenase and mitochondrial electron transport system are major sources of ROS. These findings were confirmed by subcellular fractionation studies and immunocytochemical localization of PCOs. Our observations are relevant to multiple sclerosis where decreased [GSH], mitochondrial dysfunction, excessive production of ROS, and increased [PCOs] have all been reported. Supported by NIH grants NS 47448 and IMSD GM 60201.