Selective role of glutathione in protecting human neuronal cells from dopamine-induced apoptosis

The role of glutathione and other antioxidants in dopamine-induced apoptosis has been analyzed in cultures of the human neuronal cell line NMB. Apoptosis, induced by 0.1-0.3 mM dopamine, was blocked by glutathione in a dose- and time-dependent manner. This was observed by monitoring cell morphology, cell viability, and the release of the cytosolic enzyme lactate dehydrogenase into the culture medium. L-Cysteine and N-acetylcysteine had a similar effect in protecting against dopamine neurotoxicity, but at lower concentrations than glutathione. The dopamine-induced alteration in the cell cycle profile, detected by flow cytometry (FACS), and intranucleosomal DNA fragmentation, were both blocked by glutathione. Treatment of NMB cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, increased the neurotoxic effect of, dopamine, suggesting that endogenous glutathione participates in reducing dopamine neurotoxicity. The relationship between glutathione and dopamine was further investigated by testing the effect of dopamine on the endogenous glutathione level. Dopamine decreased glutathione levels within 16-24 hr; however, this effect was preceded by a transient increase in the level of the tripeptide within the first 0.5-7 hr. Two other types of endogenous antioxidants, (+)-alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C), were tested; vitamin E (at 1-100 microgram/ml) was inactive against dopamine toxicity, whereas vitamin C had no effect at 0.05-0.2 mM, but increased dopamine toxicity at 0.5-2 mM. The results indicate that glutathione has a selective role in protecting human neural cells from the toxic effect of dopamine. This study may contribute, therefore, to a better understanding of the mechanisms underling the excessive loss of dopaminergic neurons in neurodegenerative diseases, such as Parkinsonism, and in the aging process.