Role of oxidative stress generated from the mitochondrial electron transport chain and mitochondrial glutathione status in loss of mitochondrial function and activation of transcription factor nuclear factor-kappa B: studies with isolated mitochondria and rat hepatocytes.

Abstract

Mitochondria are an important source of reactive oxygen intermediates because they are the major consumers of molecular oxygen in cells. Respiration is associated with toxicity, which is related to the activation of oxygen to reactive intermediates. The purpose of the present study was to examine the role of reduced glutathione (GSH) in the maintenance of mitochondrial functions during oxidative stress induced through selective inhibition of the complex III segment of the electron transport chain. Hydrogen peroxide monitored by the fluorescence of dichlorofluorescein increased in a time- and dose-dependent manner on incubation of mitochondria with antimycin A (AA), an inhibitor of complex III. However, blockade of complex I or II with rotenone or thenoyltrifluoroacetone, respectively, did not result in accumulation of hydrogen peroxide. Depletion of mitochondrial GSH to 10-20% of control by preincubation with diethylmaleate (0.8 mM) or ethacrynic acid (250 microM) also increased dichlorofluorescein and malondialdehyde levels and resulted in an additional (2-3-fold) increase after AA. Similar results were obtained when mitochondrial GSH depletion was produced by treatment with buthionine L-sulfoximine before mirochondria isolation. The endogenous oxidative stress induced by AA was accompanied by a moderate loss of activity of ATPase complex (77% of control) and complex IV of respiration (75% of control), which was accentuated after depletion of mitochondrial GSH (51% and 45% of control, respectively). Similar results were observed in isolated hepatocytes in which depletion of mitochondrial GSH and AA led to peroxidation and mitochondrial dysfunction. In addition, with electrophoretic mobility shift assay of the transcription factor nuclear factor-kappa B (NF-kappa B), we detected its activation in response to AA (2-3-fold). Depletion of mitochondrial GSH in hepatocytes (20% of control) led to further enhancement of NF-kappa B activation (2-4-fold), which correlated with generation of hydrogen peroxide. Thus, our results suggest that GSH protects mitochondria against the endogenous oxidative stress produced at the ubiquinone site of the electron transport chain. Mitochondrial GSH depletion potentiates oxidant-induced loss of mitochondrial functions. Oxidant stress in mitochondria can promote extramitochondrial activation of NF-kappa B and therefore may affect nuclear gene expression.