PROTECTION AGAINST GLUTAMATE-INDUCED CYTOTOXICITY IN C6 GLIAL-CELLS BY THIOL ANTIOXIDANTS

In many cell lines, glutamate cytotoxicity is known to be mediated by an inhibition of cystine transport. Because glutamate and cystine shar e the same transporter, elevated levels of extracellular glutamate com petitively inhibit cystine transport leading to depletion of intracell ular glutathione. A glutathione-depleted state impairs cellular antiox idant defenses resulting in oxidative stress. It was therefore of inte rest to investigate whether proglutathione agents, e.g., N-acetylcyste ine and lipoic acid, are able to protect against glutamate cytotoxicit y. Both lipoic acid (100 mu M-1 mM) and N-acetylcysteine (100 mu M-1 m M) completely protected C6 cells from the glutamate-induced cell death . Both agents facilitate extracellular supply of cysteine, the reduced form of cystine, that is transported into the cell by a glutamate-ins ensitive transport mechanism. Protection by lipoic acid and N-acetylcy steine corresponded with a sparing effect on cellular glutathione, whi ch is usually depleted after glutamate treatment. In the presence of L -buthionine-(S,R)-sulfoximine, a gamma-glutamylcysteine synthetase inh ibitor, low doses (<100 mu M) of lipoic acid. and N-acetylcysteine did not protect cells against glutamate-induced cytotoxicity. At higher c oncentrations (>500 mu M): however, both lipoic acid and N-acetylcyste ine provided partial protection against glutamate cytotoxicity even in glutathione one synthesis-arrested cells. These results indicate that at low concentrations the primary mechanism of protection by the thio l antioxidants was mediated by their proglutathione property rather th an direct scavenging of reactive oxygen. At higher concentrations (>50 0 mu M), a GSH-independent direct antioxidant effect of lipoic acid an d N-acetylcysteine was observed. Dichlorofluorescin fluorescence, a me asure of intracellular peroxides, increased sixfold after glutamate tr eatment of C6 cells. Lipoic acid and N-acetylcysteine treatment signif icantly lowered glutamate-induced dichlorofluorescin fluorescence comp ared with that of controls. Interestingly, ol-tocopherol (50 mu M) als o suppressed glutamate-induced dichlorofluorescin fluorescence, indica ting the peroxides detected by dichlorofluorescin were likely lipid hy droperoxides. Both thiol antioxidants, particularly lipoic acid, appea r to have remarkable therapeutic potential in protecting against neuro logical injuries involving glutamate and oxidative stress.