Antioxidants protect against glutamate-induced cytotoxicity in a neuronal cell line

The effects of reducing agents and antioxidants on L-Glutamate (Glu)-induced cytotoxicity were examined in the N18-RE-105 neuronal cell line. The cytotoxicity by Glu (1 and 10 nM) was potentiated by exposure to growth medium containing a low concentration of cystine (5-100 μM), instead of the normal medium containing 200 μM cystine. In contrast, the toxicity was suppressed by increasing the cysteine concentration to 500 to 1000 μM. Reducing agents, cystine (30-1000 μM), dithiothreitol (10-250 μM) and glutathione (GSH, 10-1000 μM) also protected the cells against the cytotoxicity of 20 mM Glu in a concentration-dependent manner. The antioxidants vitamin E (10-100 μM) [idebenone (0.-3 μM) and vinpocetine (10-100 μM)] also provided marked protection against the cytotoxicity of Glu (10 mM) or quisqualate (1 mM). Antioxidants also prevented the delayed cell death caused by lowering the concentration of cystine in the medium to 5 μM. Incubation of the cells with 10 mM Glu caused a marked decrease in cellular GSH levels. Although cysteine and dithiothreitol prevented the GSH reduction caused by Glu, antioxidants did not. The cellular levels of oxidants were assessed using 2,7-dichlorofluorescin, a probe that accumulates within cells and is converted to a fluorescent product by oxidation. Glu (10 mM) caused a marked increase in such fluorescence, whereas vitamin E and idebenone reduced markedly the number of fluorescent cells to control levels even added with 10 mM Glu. These results indicate that oxidative stress due to loss of cellular levels of GSH is one mechanism whereby Glu/quisqualate exert cytotoxicity and suggest that centrally active antioxidants may reduce neuronal damage in pathologic conditions associated with excessive Glu release.