Selective depletion of mitochondrial glutathione concentrations by (R,S)-3-hydroxy-4-pentenoate potentiates oxidative cell death

Abstract

The hepatocellular glutathione content is partitioned into a cytosolic pool, which accounts for about 85% of the cellular glutathione content, and a mitochondrial pool, which accounts for about 15% of the cellular glutathione content. Previous studies indicated that the mitochondrial glutathione pool may play a critical role in cytoprotection against xenobiotic-induced cell damage. Tests of the role of mitochondrial glutathione in cytoprotection have been hampered by the lack of agents that selectively deplete the mitochondrial glutathione pool. To test the hypothesis that mitochondrial glutathione plays a critical role in protecting against cytotoxic agents, we developed a method to deplete selectively mitochondrial glutathione concentrations. (R,S)-3-Hydroxy-4-pentenoate, an analog of (R)-3-hydroxybutanoate, caused a rapid and selective depletion of mitochondrial glutathione concentrations. Incubation of (R,S)-3-hydroxy-4-pentenoate with rat liver mitochondria or with 3-hydroxybutyrate dehydrogenase in the presence of glutathione afforded a glutathione conjugate whose chromatographic properties were identical with synthetic S-(3-oxo-4-carboxybutyl)glutathione, indicating that (R,S)-3-hydroxy-4-pentenoate was oxidized to the Michael acceptor 3-oxo-4-pentenoate, which reacts with glutathione. Exposure of rat hepatocytes to (R,S)-3-hydroxy-4-pentenoate, which was not cytotoxic and did not induce mitochondrial dysfunction, potentiated the cytotoxicity of tert-butyl hydroperoxide. These results establish the critical role of mitochondrial glutathione in cytoprotection and demonstrate and (R,S)-3-hydroxy-4-pentenoate may find utility in exploring mitochondrial glutathione homeostasis.

Highly Selective and Prolonged Depletion of Mitochondrial Glutathione in Astrocytes Markedly Increases Sensitivity to Peroxynitrite

“…There have been previous reports of selective partial decreases in the mitochondrial pool in response to experimental manipulations in vitro (Shan et al, 1993;Raza et al, 2002) or in animal models of disease (Fernandez-Checa et al, 1991;GarciaRuiz et al, 1994;Colell et al, 1998;Anderson and Sims, 2002). However, this is the first time for any cell type that such highly selective depletion of mitochondrial glutathione has been achieved.…”

Control of Mitochondrial Redox Balance and Cellular Defense against Oxidative Damage by Mitochondrial NADP+-dependent Isocitrate Dehydrogenase

“…Second, mitochondrial GSH becomes critically important against ROS-mediated damage because it not only functions as a potent antioxidant but is also required for the activities of mitochondrial glutathione peroxidase and mitochondrial phospholipid hydroperoxide glutathione peroxidase (52), which removes mitochondrial peroxides. Third, depletion of mitochondrial, but not cytosolic, GSH potentiated the oxidative cell death after treatment with tertbutyl hydroperoxide (53). Fourth, GSH, not synthesized in mitochondria, must be synthesized in the cytosol and transported into mitochondria by a specific transporter (19,54).…”

Radical-free biology of oxidative stress

“…Increased abundance of Prx3 also protected against apoptosis caused by H 2 O 2 (205). Depletion of mitochondrial GSH also potentiated peroxide accumulation and sensitized cells to cell death (8,157). Small interferring RNA (siRNA) for Grx-2 increased sensitivity to doxorubicin, whereas overexpression of mitochondrial Grx-2 inhibited cardiolipin loss and prevented apoptosis induced by doxorubicin (41,111).…”