We investigated the roles of endogenous
glutathione on
6-hydroxydopamine (6-OHDA)-induced apoptosis in human
neuroblastoma SK-N-SH cells using DNA fragmentation
enzyme-immunoassay and the
DNA dye Hoechst 33258 staining. We observed that exogenous
reduced glutathione (GSH), but not
oxidized glutathione (
GSSG), protected
6-OHDA (25 micro M)-induced apoptosis in a dose-dependent manner. Preincubation (18 h) with the
glutathione synthesis inhibitor DL-
buthionine-(S,R)-sulfoximine (BSO) significantly potentiated the toxic effects of
6-OHDA (12.5 or 25 micro M). In contrast to BSO,
N-acetylcysteine (NAC) blocked, and
L-(-)-cystine, the
glutathione precursor, significantly attenuated
6-OHDA (25 micro M)-induced apoptosis, respectively. No alterations in endogenous
glutathione concentrations were detected at 5, 15, 30, 60 min, 1 hour, 3 hours, or 6 hours after
6-OHDA (25 micro M) treatment. However, we found a 3.5-fold increase of intracellular
glutathione levels 24 hours later. On the contrary, higher concentration (100 micro M) of
6-OHDA treatment, which caused more severe cell death, showed no changes of
glutathione levels. These results suggest that delayed induction of endogenous
glutathione might play an important role in the neuroprotective mechanism against
dopamine cell death. In addition, we found that NAC might work as a beneficial catecholaminergic neuron-survival factor more efficiently than exogenous
glutathione or
L-cystine.