title: Apoptosis of alcohol-exposed human placental cytotrophoblast cells is downstream of intracellular calcium signaling.
abstract: BACKGROUND
  Apoptosis is induced by ethanol (EtOH) in human placental trophoblast cells, possibly disrupting placentation and contributing to intrauterine growth restriction in fetal alcohol spectrum disorder (FASD). EtOH induces programmed cell death in several embryonic tissues by raising intracellular Ca(2+) . Therefore, the role of Ca(2+) signaling in EtOH-induced apoptosis was examined using human first trimester cytotrophoblast cell lines, examining the hypothesis that apoptosis is dependent on intracellular Ca(2+) signaling.
  
  
  METHODS
  Using HTR-8/SVneo and SW.71 cytotrophoblast cell lines, real-time intracellular Ca(2+) concentration was monitored by fluo-4 epifluorescence microscopy and apoptosis was assessed by flow cytometry of cells fluorescently labeled for DNA fragmentation (TUNEL) and annexin V binding.
  
  
  RESULTS
  Intracellular Ca(2+) concentrations increased synchronously in all cells within 10 seconds of exposure to 50 mM EtOH, but not at lower EtOH concentrations (10 to 25 mM) incapable of inducing apoptosis. Trophoblast cells treated with inhibitors of Ca(2+) signaling (BAPTA-AM, U73122, xestospongin D, BAPTA, SKF-96365) produced no intracellular Ca(2+) transients after exposure to 50 mM EtOH and were protected from cell death induced by EtOH.
  
  
  CONCLUSIONS
  EtOH-induced apoptosis in human cytotrophoblast cells, identified by DNA fragmentation and externalized phosphatidylserine, was dependent upon Ca(2+) signaling. Both intracellular Ca(2+) mobilization and extracellular Ca(2+) influx were required, as well as phosphatidylinositol signaling. Inhibition by SKF-96365 suggests that the capacitative Ca(2+) entry mechanism that utilizes TRPC channels was activated by EtOH. Apoptosis occurs downstream of Ca(2+) signaling in trophoblasts and may contribute to placental insufficiency and poor fetal growth associated with FASD.
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University, C.S. Mott Center for Human Growth and Development, Detroit, Michigan.
authors.forename: Jay M
authors.initials: JM
authors.lastname: Bolnick
authors.forename: Rita
authors.initials: R
authors.lastname: Karana
authors.forename: Po J
authors.initials: PJ
authors.lastname: Chiang
authors.forename: Brian A
authors.initials: BA
authors.lastname: Kilburn
authors.forename: Roberto
authors.initials: R
authors.lastname: Romero
authors.forename: Michael P
authors.initials: MP
authors.lastname: Diamond
authors.forename: Susan M
authors.initials: SM
authors.lastname: Smith
authors.forename: D Randall
authors.initials: DR
authors.lastname: Armant
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D006801:Humans; D008856:Microscopy, Fluorescence; D010920:Placenta / Q000166:cytology / Q000187:drug effects*; D011247:Pregnancy; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014327:Trophoblasts / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000431:Ethanol; D002118:Calcium
keywords: Alcohol; Apoptosis; Calcium; Fetal Alcohol Spectrum Disorder; Intracellular Signaling; Intrauterine Growth Restriction; Trophoblast