Apoptosis of alcohol-exposed human placental cytotrophoblast cells is downstream of intracellular calcium signaling

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 Ca²⁺. Therefore, the role of Ca²⁺ signaling in EtOH-induced apoptosis was examined using human first trimester cytotrophoblast cell lines, examining the hypothesis that apoptosis is dependent on intracellular Ca²⁺ signaling. Methods: Using HTR-8/SVneo and SW.71 cytotrophoblast cell lines, real-time intracellular Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ signaling (BAPTA-AM, U73122, xestospongin D, BAPTA, SKF-96365) produced no intracellular Ca²⁺ 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 Ca²⁺ signaling. Both intracellular Ca²⁺ mobilization and extracellular Ca²⁺ influx were required, as well as phosphatidylinositol signaling. Inhibition by SKF-96365 suggests that the capacitative Ca²⁺ entry mechanism that utilizes TRPC channels was activated by EtOH. Apoptosis occurs downstream of Ca²⁺ signaling in trophoblasts and may contribute to placental insufficiency and poor fetal growth associated with FASD.