Ethanol-induced cephalic apoptosis requires phospholipase C-dependent intracellular calcium signaling.
BACKGROUND
Although the ability of ethanol to elicit neural crest cell apoptosis is well documented, the initial target of ethanol in these cells, and the biochemical pathway leading to their apoptosis, have yet to be determined. Recent work in preimplantation mouse embryos demonstrates that ethanol induces a phospholipase-C (PLC)-dependent calcium transient that mediates ethanol's effects. We tested whether a similar effect on calcium and PLC is involved in ethanol-induced neural crest apoptosis.


METHODS
Chicken embryos were collected and loaded with Fluo-3-AM to assess the effects of ethanol on intracellular calcium levels. Pharmacological agents were used to determine the sources and mechanism of intracellular calcium increases. In separate experiments, embryos were treated in ovo with pharmacological modulators of calcium signaling prior to ethanol exposure, and resulting levels of cell death were assessed by using the vital dye acridine orange.


RESULTS
Ethanol exposure caused a localized increase in intracellular calcium levels in embryonic neural folds within 15 sec of ethanol exposure. Ethanol-induced apoptosis was specifically blocked by chelation of intracellular calcium before ethanol exposure. Pretreatment with the PLC inhibitor U73122 blocked ethanol-induced apoptosis as well as the intracellular calcium transient. Depletion of extracellular calcium resulted in a partial block of ethanol-induced apoptosis.


CONCLUSIONS
Ethanol exposure alters calcium signaling within the neurulation-stage chicken embryo in a PLC-dependent manner. Increases in intracellular calcium and PLC activity are necessary for ethanol's induction of apoptosis within cephalic populations. These effects likely represent an early and crucial event in the pathway leading to ethanol-induced cell death.
Debelak-Kragtorp
Katherine A
KA
Program in Molecular and Environmental Toxicology, University of Wisconsin-Madison, 53706, USA.
Armant
D Randall
DR
Smith
Susan M
SM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002642:Chick Embryo; D000431:Ethanol / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000187:drug effects / Q000201:enzymology; D009432:Neural Crest / Q000187:drug effects* / Q000196:embryology / Q000201:enzymology; D010738:Type C Phospholipases / Q000378:metabolism*
D000431:Ethanol; D010738:Type C Phospholipases