| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 102 === Chronic ethanol exposure-associated oxidative stress causes various degrees of brain damage among individuals depending on their exposure to other environmental factors, such as organic environmental pollutants that activate aryl hydrocarbon receptors (AhR). This study was to investigate how the combination of subchronic ethanol ingestion and AhR ligand exposure regulates mitochondrial enzymes, the oxidative stress-associated cytochrome P450 2E1 (CYP2E1) and CYP1A1, and the anti-oxidative Cu/Zn-superoxide dismutase (SOD1) in cerebral cortical neurons. In this study, primary cultured cortical neurons were prepared from embryonic day 17 Sprague Dawley fetal rats, and cultured neurons at 9 days in vitro (9 DIV) were used in our study. The cultured neurons were subjected to subchronic ethanol treatment, i.e. 80mM ethanol for 3 days, followed by incubating with or without AhR agonists, a natural ligand tryptophan photoproduct 6-formylindolo [3,2-b] carbazole (FICZ, 20nM) or the environmental ligand 2,3,7,8-tetrachlorodibenzodioxin (TCDD, 20nM) for 24hr. Immunofluorescent staining and WST-1 assay indicated that subchronic ethanol and AhR ligand induced neuronal loss. Subchronic ethanol increased CYP2E1 protein expression as examined by Western blotting in cortical neurons, and the effect was suppressed by the AhR agonist application. The induction of CYP1A1, an AhR target gene, was enhanced when AhR agonists were applied under subchronic ethanol treatment. In contrast, subchronic ethanol does not affect the AhR ligand-induced AhR protein and mRNA downregulation. The anti-oxidative enzyme SOD1 was reduced under subchronic ethanol exposure with or without AhR ligands, whereas AhR ligands can induce SOD1 mRNA but the effect was diminished under subchronic ethanol treatment. Reciprocally, we found that subchronic ethanol - AhR agonist exposure can increase oxidative stress as determined by NADPH oxidase activity assay. Next, we used AhR antagonist TMF to block AhR activation, and found that TMF suppressed SOD1 expression and increased oxidative stress in the ethanol-free condition, but can enhance SOD1 expression without inducing oxidative stress in neurons under subchronic ethanol exposure. Finally, we established a subchronic binge ethanol animal model, and found that SOD1 and CYP1A1 mRNA expressions were both increased in the rat cerebral cortex and hippocampus, whereas the SOD1 mRNA in the cerebellum was decreased. Together, our data tend to suggest a cross-talk between the subchronic ethanol ingestion and AhR activation reciprocally enhances or attenuates their own primary effects on the expression of CYP2E1, CYP1A1, and SOD1, which may subsequently regulate oxidative stress in cortical neurons and alcoholic brains. Information obtained from this preliminary study may provide clues for the impact and mechanism of environmental confounding factors on the alcoholism-associated neurological deficits.
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