title: Nicotine-induced embryonic malformations mediated by apoptosis from increasing intracellular calcium and oxidative stress.
abstract: BACKGROUND
  Tobacco smoking by women during pregnancy increases the risk of congenital birth defects in the infants. Among the smoke products, nicotine is believed to be the major teratogenic factor that perturbs embryonic development. However, the role of nicotine in embryonic malformations has not been addressed, and the mechanisms by which nicotine affects embryonic development remain to be delineated.
  
  
  METHODS
  To investigate the effects of nicotine on early embryogenesis, murine embryos at embryonic day (E) 8.5 were dissected out of the uteri, cultured in a roller bottle system, and treated with nicotine (0.6-6 microM) or vehicle. Embryonic morphogenesis and growth were examined in terms of structural morphology and crown/rump length, respectively. Programmed cell death (apoptosis) was assessed using LysoTracker Red staining of whole mount embryos and TUNEL assay of tissue sections. Changes in intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) production were assessed using fluorescent dyes (Flu-4, AM; H2DCFDA, respectively) under a confocal microscope. To further investigate the role of intracellular calcium and ROS in nicotine-induced embryopathy, embryos were treated with BAPTA-AM (2 microM) to inhibit [Ca2+]i elevation and ascorbic acid (vitamin C; 100 microg/ml) to scavenge ROS in presence of nicotine (6 microM).
  
  
  RESULTS
  The embryos treated with nicotine in 3-6 microM were smaller than those treated with vehicle. Most of the embryos had open neural tube in the anterior (brain) regions. The embryos treated with 6 microM nicotine also exhibited severe defects in the posterior trunk, resembling caudal dysplasia. Excessive apoptosis was observed in the deformed structures. Significant increases in [Ca2+]i and ROS were seen in the tissues that had higher levels of apoptosis. Furthermore, inhibition of [Ca2+]i and scavenging of ROS significantly reduced embryonic malformation and apoptotic rates in the embryos.
  
  
  CONCLUSIONS
  Nicotine affects embryonic development in a concentration-dependent manner. The nicotine-induced embryonic malformations are, in part, a result of excessive cell death. Nicotine increases [Ca2+]i and ROS level, which play a role in nicotine-induced embryonic apoptosis and malformations. These studies identify the molecular pathway of nicotine action in embryonic apoptosis and malformations, and provide a promising approach for ameliorating the teratogenic effects of nicotine.
authors.affiliation: Department of Obstetrics and Gynecology, The Arkansas Center for Birth Defects Research and Prevention, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. zzhao@uams.edu
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Zhao
authors.forename: E Albert
authors.initials: EA
authors.lastname: Reece
mesh_terms: D000014:Abnormalities, Drug-Induced / Q000209:etiology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000473:pathology; D005145:Face / Q000196:embryology; D005260:Female; D005317:Fetal Growth Retardation / Q000209:etiology; D016166:Free Radical Scavengers / Q000494:pharmacology; D005731:Ganglionic Stimulants / Q000633:toxicity*; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D009436:Neural Tube Defects / Q000139:chemically induced / Q000196:embryology; D009538:Nicotine / Q000633:toxicity*; D018384:Oxidative Stress; D011247:Pregnancy; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D016166:Free Radical Scavengers; D005731:Ganglionic Stimulants; D017382:Reactive Oxygen Species; D009538:Nicotine; D002118:Calcium