Rapid Induction of Apoptosis in Gastrulating Mouse Embryos by Ethanol and Its Prevention by HB‐EGF

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Abstract
Background:Ethanol exposure during gastrulation and early neurulation induces apoptosis within certain embryonic cell populations, leading to craniofacial and neurological defects. There is currently little information about the initial kinetics of ethanol‐induced apoptosis, and interest in the ability of endogenous survival factors to moderate apoptosis is growing. Ethanol alters intracellular signaling, leading to cell death in chick embryos, suggesting that apoptosis could occur rapidly and that signaling pathways activated by survival factors might reduce apoptosis.Methods:Pregnant mice were intubated with 1, 2, or 4 g/kg ethanol on day 7.5 of embryogenesis (E7.5) 1, 3, or 6, hours before harvesting gastrulation‐stage embryos. Control animals received maltose/dextran. Blood alcohol concentrations (BAC) were determined by gas chromatography. E7.5 embryos isolated from untreated dams were cultured in vitro for 1 or 3 hr with 0 or 400 mg% ethanol and 0 or 5 nM heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF). Apoptosis was quantified using fluorescence microscopy to detect annexin V binding and DNA fragmentation [terminal deoxynucleotidyl transferase‐mediated dUTP‐Xnickendlabeling (TUNEL)] in whole‐mount or sectioned embryos.Results:Both annexin V binding and TUNEL were elevated (pppConclusions:Ethanol rapidly produced apoptosis in gastrulation‐stage embryos, consistent with induction by intracellular signaling. The ethanol‐induced apoptotic pathway was blocked by the endogenous survival factor, HB‐EGF. Differences in the expression of survival factors within individual embryos could be partly responsible for variations in the teratogenic effects of ethanol among offspring exposed prenatally.

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