Differential effect of ethanol on PC12 cell death

Our goal was to examine the effects of ethanol on cell death using rat pheochromocytoma (PC12) cells as a neuronal model. Withdrawal of serum for 24 hr increased the number of PC12 cells labeled with ethidium homodimer indicating an increase in cell death. Inclusion of 50 mM ethanol during the serum deprivation further increased the amount of ethidium fluorescence by 39%. Although reducing the serum concentration from the usual 15 to 4% did not alter cellular viability, a significant increase in the amount of ethidium fluorescence was observed in PC12 cells incubated for 24 hr in the presence of 4% serum and 150 mM ethanol. No change in viability was observed in cells exposed to either 150 mM ethanol in the presence of 15% serum or 50 mM ethanol in the presence of 4% serum. Inclusion of ethanol during serum deprivation increased the amount of soluble DNA found in the 15,000 x g supernatant. Similarly, using the terminal deoxynucleotidyl transferase dUTP nick-end labeling method to visualize DNA fragmentation in situ, ethanol caused a 213% increase in the number of PC12 cells labeled during serum withdrawal. Agarose gel electrophoresis of the DNA isolated from cells maintained in the absence of serum yielded the classical DNA laddering pattern of 180 to 200 bp fragments suggestive of apoptosis. Ethanol caused a concentration-dependent increase in the amount of DNA laddering in cells deprived of serum. Furthermore, ethanol significantly potentiated the DNA laddering of cells maintained in 4% serum. In contrast to the ethanol-induced increase in cell death when serum factors were reduced or withdrawn, 150 mM ethanol lowered by 34% the number of ethidium-labeled PC12 cells observed after a 30-min exposure to 2 mM H2O2. Similarly, agarose gel electrophoresis of the DNA from H2O2-treated cells did not display DNA laddering. This study demonstrates that: 1) ethanol antagonizes the trophic action of serum factors; 2) pharmacologically relevant ethanol concentrations significantly potentiate apoptosis after serum withdrawal and 3) this enhancement appears specific for apoptosis.