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

Background: The central nervous system is particularly sensitive to the cyt otoxic effect of ethanol. In vivo and in vitro studies indicate that ethano l decreases cell proliferation in a number of cells types, including neuron s and glial cells in the central nervous system. The cellular mechanisms in volved in ethanol-induced cell toxicity, however, are unclear. In this stud y, we examined the effect of ethanol on tumor necrosis factor-alpha (TNF al pha)-induced cell death in a homogeneous population of cultured rat astrocy tes. Methods: Flow cytometric and 3-(4,5-dimethylthiazol-2-yl) -2,5-dipbenytetra zolium bromide (MTT) dye reduction analyses were performed on cultured rat astrocytes to determine the effect of alcohol on TNF alpha-induced cell dea th. Results: Flow cytometric analysis revealed that, in quiescent astrocytes, h igh concentrations of ethanol were required to increase DNA fragmentation a nd decrease cell viability. Preexposure of astrocytes to low concentrations of ethanol (10 to 50 mM). however, increased the sensitivity of astrocytes to TNF alpha with low TNF alpha concentrations (25 to 50 ng/ml) resulting in increased DNA fragmentation. Furthermore, MTT dye reduction analysis rev ealed that exposure of astrocytes to 5 mM ethanol was sufficient to increas e the susceptibility of astrocytes to the cytotoxic effect of ethanol. In a number of cell types, TNF alpha receptor binding results in the activation of specific signal transduction cascades, including the hydrolysis of sphi ngomyelin to ceramide. We show that preexposure of astrocytes to a low conc entration of ethanol increased the sensitivity of astrocytes to sphingomyel inase, and C-2-ceramide resulting in increased DNA fragmentation and decrea sed cell viability. More importantly, astrocytes prepared from rats exposed to ethanol prenatally showed increased susceptibility to TNF alpha-induced cell death. Conclusions: These studies suggest that ethanol increases the susceptibilit y of astrocytes to TNF alpha-induced cell death by shifting the balance of sphingolipid metabolism in favor of a pathway that increases the susceptibi lity of astrocytes to the cytotoxic effect of TNF alpha.