Studies on the Antioxidative and Cytotoxic Actions of Epigallocatechin Gallate in Murine Cerebral Cortical Cell Cultures

OBJECTIVE: Epigallocatethin gallate(EGCG) is a major green tea polyphenol and is known to have potent antioxidative and antiproliferative actions. This study is performed to investigate the antioxidative effect of EGCG on the various oxidative insults in mouse cerebral cortical cell cultures. METHODS: Mixed cortical cell cultures containing both neuron and glia prepared by plating fetal mice cortical cells on to an established glia of 24 well vessels. At 13-15 days in vitro, oxidative neuronal deaths were induced by the addition of oxidants into the cortical cultures. Iron ion(FeCl2), copper ion(CuCl2), sodium nitroprusside(SNP) and buthionine sulfoximine(BSO, a glutathione depletor) were used as oxidants. Cell death was assessed by LDH assay after microscopic examination. RESULTS: All four oxidants induced neuronal cell death associated with cell body swelling, which was markedly inhibited by Trolox(100muM), a vitamin E analog. EGCG(1-10muM) markedly inhibited the neuronal cell death induced by 20muM CuCl2, 1muM SNP, or 1mM BSO. Unexpectedly the neuronal cell death induced by 20muM FeCl2 was augmented by treatment with 1 or 3muM EGCG. EGCG itself induced concentration- and exposure time-dependent cell death at more than 30muM concentrations. EGCG(30, 100muM) injured not only neuronal cells but glial cells after 48 hour exposure. The EGCG-induced cytotoxicity was partially inhibited by protein synthesis inhibitors, cycloheximide(0.1 or 1mug/ml) and emetine (1mug/ml) or high potassium media(10 or 25mM) but was not affected by Trolox. CONCLUSION: These results suggest that the dual antioxidative-cytotoxic actions of EGCG are concentration-dependent and that the antioxidative aciton depends on the kind of oxidative insults, and that the EGCG-induced cytotoxicity be relevant to protein synthesis and/or membrane depolarization.

Effect of Trolox, Cycloheximide or MK-801 on the Neuronal Cell Death Induced by FeCl2, Buthionine Sulfoximine or KCN in Primary Murine Mixed Cortical Cell Culture

Oxidative stress is known to be a major neuropathologic mechanism in chronic neurodegenerative disorders as well as in stroke, trauma and epilepsy, and many kinds of oxidative insults induce neuronal injury. The purpose of this study was to examine the temporal effects of trolox(TLX; water and lipid soluble vitamin E analog), cycloheximide(CHX; protein synthesis inhibitor) and MK-801(NMDA receptor antagonist) on neuronal death indu-ced by different kinds of oxidative insults in primary murine mixed cortical cell culture(14-16 days in vitro), and to gain information on the mechanisms underlying oxidative cell death. As oxidative insults, the authors used iron(FeCl2), buth-ionine sulfoximine(BSO; glutathione depletor) and potassium cyanide(KCN; ATP depletor). Cell death was assessed by measurement of LDH efflux to bathing media at the end of exposure. All three agents induced neuronal cell death associated with cell body swelling. FeCl2(30nM-1mM) induced conce-ntration- and exposure time-dependent neurotoxicity, while BSO(10nM-3mM) showed little neurotoxicity at the end of 24 hrs exposure, but marked neuronal cell death at the end of 48 hrs; at concentrations of over 100uM of BSO neurotoxicity reached a plateau. KCN(0.1mM-1mM) also showed dose-dependent neurotoxicity. TLX(100nM) did not affect the neurotoxicity induced by KCN(1mM) but almost completely block BSO(1mM)- or FeCl2(100, 300nM)- induced neuronal cell death. CHX(1ng/ml) significantly attenuated BSO-induced cell death but did not protect against KCN(1mM)-induced cell death. CHX treatment, on the other hand, significantly potentiated FeCl2(100 or 300nM)-induced death. MK-801(10nM) markedly inhibited KCN-induced cell death but had no effect on FeCl2-induced death. MK-801 also significantly attenuated BSO-induced neurotoxicty after exposure for 48hrs but this protective effect disappeared at the end of 72hr. These results suggest that protein synthesis as well as lipid peroxidation of cell membrane may involve oxidative neuronal injury and that one oxidative agent may induce various cell death processes.