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.

Oxidative injury in multiple sclerosis cerebellar grey matter

“…Imbalances in the cellular redox homeostasis (increased ROS production alongside a lack in hydrogen peroxide detoxifying enzyme activity) may lead to an excess of hydrogen peroxide and the subsequent formation of 4-HNE and/or malondialdehyde as evident in this study. In support of this theory, in vitro studies have directly correlated decreased glutathione levels and GPX activity with increased lipid peroxidation and protein carbonylation (Bizzozero et al, 2006;Bizzozero et al, 2007). Our findings may therefore suggest that in MS the lack in activity of hydrogen peroxide detoxifying 11 enzymes during the inflammatory insult may conceivably play a pathogenic role in the accumulation of lipid peroxidation, oxidative stress and myelin injury in MS GM.…”

α-Lipoic acid (LA) enantiomers protect SH-SY5Y cells against glutathione depletion

“…In experimental animal studies, acute depletion of glutathione caused extensive carbonylation of brain proteins (Bizzozero et al, 2006) and morphological changes of dopaminergic neurons in the nigrostriatal pathway that mimic alterations observed in the brains of aged animals (Andersen et al, 1996). It is, therefore, critical to find pathways which can improve neuronal survival / activity in the glutathione-depleted condition.…”

Glutathione depletion causes a JNK and p38MAPK-mediated increase in expression of cystathionine-γ-lyase and upregulation of the transsulfuration pathway in C6 glioma cells

“…In the second phase of the study, we addressed the question of whether pre-treatment of the cells with DEM would also lead to increased expression of cystathionine-y-lyase. DEM causes direct depletion of reduced GSH (Bizzozero et al, 2006) independently of the concentration of cysteine and enabled us to distinguish between lowered intracellular cysteine or GSH as the trigger to increase expression of cystathionine-γ-lyase. The outcome was the same, regardless of whether the drop in GSH was a result of gliotoxins or DEM and we conclude that transulfuration provides a compensatory mechanism that is upregulated in response to depletion of GSH or oxidative stress in C6 cells.…”