title: Acute depletion of reduced glutathione causes extensive carbonylation of rat brain proteins.
abstract: 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[2-chloroethyl]-1-nitrosourea (BCNU) and diethyl maleate (DEM), and protein carbonylation was assessed on Western blots after derivatization with dinitrophenyl hydrazine. Incubation with 1 mM BCNU or 10 mM DEM for 2 hr decreased GSH levels by > 70%. Under these conditions the carbonylation of several proteins (40-120 kDa) increased by 2-3 fold. Isolation of carbonylated proteins showed that augmented PCOs represents a rise in the amount of oxidized protein. The iron chelator deferoxamine, the superoxide scavenger rutin and the H2O2 quencher dimethylthiourea all prevented DEM-induced protein carbonylation and lipid peroxidation (TBARS), indicating that the underlying mechanism involves the iron-catalyzed generation of hydroxyl radicals from H(2)O(2) (Fenton reaction). Inhibition of catalase activity with sodium azide and aminotriazole, and glutathione peroxidase activity with mercaptosuccinic acid did not increase PCOs or TBARS, suggesting that increased production of reactive oxygen species (ROS) rather than compromised cellular antioxidant defenses is the cause for the accumulation of H2O2 after GSH depletion. PCO formation was not affected by the xanthine oxidase inhibitor oxypurinol but it was reduced by SKF-525A and carbonyl cyanide 3-chlorophenylhydrazone, indicating that the microsomal monooxygenase system and the mitochondrial electron transport system are the major sources of ROS. Consistent with these findings, subcellular fractionation studies showed that mitochondria and synaptosomes are the major PCO-containing organelles. These results were also supported by the anatomic distribution of PCOs in brain. Our observations may be important in the context of multiple sclerosis where decreased GSH, mitochondrial dysfunction, excessive production of ROS, and increased protein carbonylation have all been reported.
authors.affiliation: Department of Cell Biology and Physiology, University of New Mexico-Health Sciences Center, Albuquerque, New Mexico 87131-5218, USA. obizzozero@salud.unm.edu
authors.forename: Oscar A
authors.initials: OA
authors.lastname: Bizzozero
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Ziegler
authors.forename: Gisela
authors.initials: G
authors.lastname: De Jesus
authors.forename: Federico
authors.initials: F
authors.lastname: Bolognani
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000172:deficiency* / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000037:antagonists & inhibitors; D017665:Hydroxyl Radical / Q000378:metabolism; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D015227:Lipid Peroxidation / Q000187:drug effects; D008293:Malates / Q000494:pharmacology; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010117:Oxypurinol / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D013347:Subcellular Fractions / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D008293:Malates; D009419:Nerve Tissue Proteins; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D014475:Uncoupling Agents; D017665:Hydroxyl Radical; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C058705:diethyl malate; D002374:Catalase; D005979:Glutathione Peroxidase; D010117:Oxypurinol; D005978:Glutathione; D003676:Deferoxamine; D002330:Carmustine