Roles of endogenous glutathione levels on 6-hydroxydopamine-induced apoptotic neuronal cell death in human neuroblastoma SK-N-SH cells.
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.
Shimizu
Eiji
E
Department of Psychiatry (K2), Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuouku, Chiba 260-8670, Japan. eiji@psy3.m.chiba-u.ac.jp
Hashimoto
Kenji
K
Komatsu
Naoya
N
Iyo
Masaomi
M
D000111:Acetylcysteine / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles; D001932:Brain Neoplasms / Q000473:pathology*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D003553:Cystine / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D009447:Neuroblastoma / Q000473:pathology*; D009474:Neurons / Q000187:drug effects*; D016627:Oxidopamine / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D013565:Sympatholytics / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D014407:Tumor Cells, Cultured
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers; D013565:Sympatholytics; D003553:Cystine; D019328:Buthionine Sulfoximine; D016627:Oxidopamine; D005978:Glutathione; C017807:bisbenzimide ethoxide trihydrochloride; D000111:Acetylcysteine