No abstract

Abstract:We examined the effects of oxidative stress on rat cultured mesencephalic neurons and glial cells. Glial cells were more resistant to 6-hydroxydopamine (6-OHDA) and H 2 O 2 toxicity than neurons. In glial cells, incubation with 6-OHDA and H 2 O 2 induced a significant increase in the expression of ␥-glutamylcysteine synthetase (the rate-limiting enzyme in glutathione synthesis) mRNA, which correlated well with increased TPA-response element (TRE)-binding activity. Furthermore, a subsequent elevation in cellular total glutathione content was also observed. In neurons, both agents decreased TRE-binding activity, and these cells failed to up-regulate the glutathione synthesis. We also examined the mechanisms of the neuroprotective effects of glial cells using a glia conditioned medium. Neurons maintained in glia conditioned medium up-regulated the level of TRE-binding activity, ␥-glutamylcysteine synthetase mRNA expression, and total glutathione content in response to 6-OHDA or H 2 O 2 , and became more resistant to both agents than cells maintained in a normal medium. Neurons maintained in normal medium failed to up-regulate the glutathione synthesis. Our results suggest that transcriptional up-regulation of glutathione synthesis in glial cell appears to mediate brain glial cell resistance against oxidative stress, and that glial cells protect neurons via transcriptional up-regulation of the antioxidant system.

We examined the effect of pretreatment with EPC-K1, a potent hydroxyl radical scavenger, on 6-hydroxydopamine (6-OHDA)-induced reduction of dopamine (DA) and its metabolites in the mouse striatum. EPC-K1 was mixed with diet (0.2%, wt/wt) for 1 or 2 weeks, and then 6-OHDA (60 microg in 2 microl of saline solution) was injected intracereberoventricularly. Mice continued to be fed EPC-K1-containing diet for another one week before they were sacrificed. The concentrations of DA and its metabolites in the striatum were measured by high performance liquid chromatography. 6-OHDA reduced the level of DA and its metabolites in the striatum. Pretreatment with EPC-K1 for 2 weeks, but not for 1 week, abrogated the neurotoxic effect of 6-OHDA on striatal concentrations of DA and its metabolites. Measurement of striatal concentrations of thiobarbituric acid reactive substances, glutathione, and malonaldehyde plus 4-hydroxynonenal, and the activities of superoxide dismutase and catalase in EPC-K1 treated mice showed an increase in catalase activity after 2 weeks of such treatment. No other changes in anti-oxidants levels were noted. Our results suggest that EPC-K1 counteracts the neurotoxicity of 6-OHDA by increasing catalase activities.

Free radicals are involved in neuronal damage. Bifemelane hydrochloride has been reported to protect neural tissues against ischemic damage and age-related neurodegeneration. We examined the protective effects of bifemelane HCl and the relation between its effectiveness and free radical formation in hydrogen peroxide (H2O2)-induced cytotoxicity using cultured rat neuroblastoma cell line (B50). Cytotoxicity was examined by using the lactate dehydrogenase (LDH) assay and cell viability by the WST-1 assay. H2O2 reduced the survival of B50 cells in a dose-dependent manner, and treatment of these cells with 75 microM or 100 microM H2O2 reduced their viability by 50% relative to the control group. B50 cells were treated with 5 or 10 microM bifemelane for 2 days followed by treatment with 75 microM or 100 microM H2O2. H2O2 cytotoxicity was reduced by pretreatment with bifemelane. We also examined the effect of bifemelane on lipid peroxide formation in B50 cells using thiobarbituric acid reactive substances assay. Pretreatment of B50 cells with 10 microM bifemelane for 2 days reduced lipid peroxide formation to approximately 54% of the control group. Our results suggest that bifemelane hydrochloride provides a protective effect against H2O2 cytotoxicity partly due to its anti-oxidative properties.