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 seru m for 24 hr increased the number of PC12 cells labeled with ethidium h omodimer indicating an increase in cell death. Inclusion of 50 mM etha nol during the serum deprivation further increased the amount of ethid ium fluorescence by 39%, Although reducing the serum concentration fro m the usual 15 to 4% did not alter cellular viability, a significant i ncrease in the amount of ethidium fluorescence was observed in PC12 ce lls 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 m M ethanol in the presence of 15% serum or 50 mM ethanol in the presenc e of 4% serum. Inclusion of ethanol during serum deprivation increased the amount of soluble DNA found in the 15,000 x g supernatant. Simila rly, using the terminal deoxynucleotidyl transferase dUTP nick-end lab eling method to visualize DNA fragmentation in situ, ethanol caused a 213% increase in the number of PC12 cells labeled during serum withdra wal. Agarose gel electrophoresis of the DNA isolated from cells mainta ined in the absence of serum yielded the classical DNA laddering patte rn of 180 to 200 bp fragments suggestive of apoptosis. Ethanol caused a concentration-dependent increase in the amount of DNA laddering in c ells 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 In contrast to the ethanol-induced increase in cell death when serum factors were reduce d or withdrawn, 150 mM ethanol lowered by 34% the number of ethidium-l abeled PC12 cells observed after a 30-min exposure to 2 mM H2O2. Simil arly, agarose gel electrophoresis of the DNA from H2O2-treated cells d id 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 af ter serum withdrawal and 3) this enhancement appears specific for apop tosis.