Ethanol increases the neurotoxic effect of tumor necrosis factor-α in cultured rat astrocytes

Background: The central nervous system is particularly sensitive to the cytotoxic effect of ethanol. In vivo and in vitro studies indicate that ethanol decreases cell proliferation in a number of cells types, including neurons and glial cells in the central nervous system. The cellular mechanisms involved in ethanol-induced cell toxicity, however, are unclear. In this study, we examined the effect of ethanol on tumor necrosis factor-α (TNFα)-induced cell death in a homogeneous population of cultured rat astrocytes. Methods: Flow cytometric and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) dye reduction analyses were performed on cultured rat astrocytes to determine the effect of alcohol on TNFα-induced cell death. Results: Flow cytometric analysis revealed that, in quiescent astrocytes, high concentrations of ethanol were required to increase DNA fragmentation and decrease cell viability. Preexposure of astrocytes to low concentrations of ethanol (10 to 50 mM), however, increased the sensitivity of astrocytes to TNFα with low TNFα concentrations (25 to 50 ng/ml) resulting in increased DNA fragmentation. Furthermore, MTT dye reduction analysis revealed that exposure of astrocytes to 5 mM ethanol was sufficient to increase the susceptibility of astrocytes to the cytotoxic effect of ethanol. In a number of cell types, TNFα receptor binding results in the activation of specific signal transduction cascades, including the hydrolysis of sphingomyclin to ceramide. We show that preexposure of astrocytes to a low concentration of ethanol increased the sensitivity of astrocytes to sphingomyelinase, and C2-ceramide resulting in increased DNA fragmentation and decreased cell viability. More importantly, astrocytes prepared from rats exposed to ethanol prenatally showed increased susceptibility to TNFα-induced cell death. Conclusions: These studies suggest that ethanol increases the susceptibility of astrocytes to TNFα-induced cell death by shifting the balance of sphingolipid metabolism in favor of a pathway that increases the susceptibility of astrocytes to the cytotoxic effect of TNFα.