Characterization of the role of glutahione in repin-induced mitochondrial dysfunction, oxidative stress and dopaminergic neurotoxicity in rat pheochromocytoma (PC12) cells

Repin, a major constituent in extracts of the plant Centaurea repens is thought to be the active principal responsible for the development of equine nigropallidal enctphalomalacia (ENE), a fatal Parkinson-like neurodegenerative disorder in horses. Although the exact mechanism by which ingestion of this weed causes ENE is uncertain, a limited body of experimental evidence suggests a critical role for the glutathione redox system. In the present study, the mechanism of repin neurotoxicity was examined in PC12 cells with a focus on determining the role of glutathione (GSH) in repininduced mitochondrial dysfunction, oxidative stress and dopaminergic toxicity. The results demonstrate that repin reduced both cellular GSH levels and mitochondrial function in a manner that was time- and concentration-dependent. The repin-induced changes in GSH levels were found to precede the changes in mitochondrial function. Depletion of GSH with a potent GSH depletor (ethacrynic acid (EA)) and a GSH synthesis inhibitor (buthionine sulfoximine (BSO)) prior to repin treatment enhanced the repin-induced mitochondrial change. In addition, repin caused a concentration-dependent decrease in cellular dopamine levels in NGF-differentiated PC12 cells. Increases in intracellular GSH levels induced by pre-treatment with reducing agents (N-acetyl-L-cysteine or reduced glutathione) completely protected the cells from repin-induced mitochondrial and dopaminergic toxicity. Antioxidants, coenzyme-Q and ascorbic acid completely blocked repin-induced dopaminergic toxicity. These data suggest that GSH plays a critical role in repin-induced neurotoxicity and that the maintenance of neuronal redox status may prove to be a useful strategy for the prevention and/or treatment of ENE. The results support the view that GSH depletion, leading to oxidative damage and subsequent mitochondrial dysfunction, may serve as a trigger for neuronal cell death. (C) 2004 Elsevier Inc. All rights reserved.