title: Rapid induction of apoptosis in gastrulating mouse embryos by ethanol and its prevention by HB-EGF.
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-X nick end labeling (TUNEL)] in whole-mount or sectioned embryos.
  
  
  RESULTS
  Both annexin V binding and TUNEL were elevated (p < 0.05) in embryos exposed in utero to 1 g/kg ethanol for 3 hours, increasing linearly with time and ethanol concentration. Apoptosis increased (p < 0.05) in all germ cell layers. Mice treated with 4 g/kg sustained BAC of 400 mg% for nearly 3 hours, significantly increasing apoptosis within the first hour. Cultured embryos exposed to 400 mg% ethanol displayed 2- to 3-fold more TUNEL than vehicle-treated embryos (p < 0.05); however, exogenous HB-EGF prevented apoptosis.
  
  
  CONCLUSIONS
  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.
authors.affiliation: C.S. Mott Center for Human Growth and Development, Department of Obstetrics & Gynecology, Wayne State University, Detroit, Michigan 48201-1415, USA.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Kilburn
authors.forename: Po Jen
authors.initials: PJ
authors.lastname: Chiang
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: George R
authors.initials: GR
authors.lastname: Flentke
authors.forename: Susan M
authors.initials: SM
authors.lastname: Smith
authors.forename: D Randall
authors.initials: DR
authors.lastname: Armant
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D047108:Embryonic Development / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D000431:Ethanol / Q000633:toxicity*; D005260:Female; D063647:Fetal Alcohol Spectrum Disorders / Q000473:pathology*; D005775:Gastrula / Q000187:drug effects* / Q000473:pathology; D066257:Heparin-binding EGF-like Growth Factor; D020287:In Situ Nick-End Labeling; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D011247:Pregnancy; D018000:Receptors, Peptide / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D018000:Receptors, Peptide; C080707:annexin V receptor; D000431:Ethanol; D004815:Epidermal Growth Factor