Mechanism of ethanol enhancement of apoptosis and caspase activation in serum-deprived PC12 cells.
Neuronal death is one of the most prominent consequences of alcohol exposure during development. Ethanol-induced neuronal death appears to involve apoptosis. The objective of the present study was to characterize the effect of ethanol on neuronal cell viability and to determine the mechanism by which ethanol enhances apoptosis in neural cells. For these studies the rat pheochromocytoma (PC12) cells were used. PC12 cells were incubated for 24 h in the presence or absence of 100 mM ethanol. Apoptosis was induced by serum withdrawal. Ethanol in the presence of serum-containing media did not alter cell viability, while incubation of PC12 cells in serum-free media resulted in a significant increase in cell death that was further significantly increased by 35% in cells exposed to ethanol. The temporal response of the PC12 cells to serum withdrawal was studied over a period of 22 h. At least 18 h of ethanol exposure was necessary to observe a significant increase in death for cells incubated in serum-free media. An increase in the caspase-3 activity in PC12 cells deprived of serum was observed that was further increased by ethanol exposure. This increase of caspase-3 activity was correlated with an enhancement of caspase-9 activity. Ethanol exposure increased the amount of cytosolic cytochrome c in PC12 cells incubated in serum-free media but did not alter the level of cytochrome c in cells incubated in serum. Finally, a 26% increase was observed in the number of cells with depolarized mitochondria due to ethanol treatment. The present study implicates an increase in the mitochondrial outer membrane permeability as a potential mechanism of enhancement of apoptosis in serum-deprived PC12 cells by ethanol.
Krzyzanski
Wojciech
W
Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States.
Oberdoester
Jan
J
Rabin
Richard A
RA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D016895:Culture Media, Serum-Free; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D000431:Ethanol / Q000633:toxicity*; D005434:Flow Cytometry; D015151:Immunoblotting; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016716:PC12 Cells; D051381:Rats
D016895:Culture Media, Serum-Free; D000431:Ethanol; D045304:Cytochromes c; D020169:Caspases