title: Altered gene expression in neural crest cells exposed to ethanol in vitro.
abstract: OBJECTIVE
  to characterize and compare ethanol-induced changes of gene expression in cells from the cranial (cNCC) and trunk (tNCC) portion of the neural crest cell (NCC) population of day-10 rat embryos.
  
  
  BACKGROUND
  previous work has suggested that ethanol-induced embryonic maldevelopment is associated with oxidative stress, and, in particular, that ethanol-induced anomalies of the facial skeleton and heart are associated with disturbed development of the cNCC. We studied alterations of mRNA levels of genes involved in apoptosis, oxidative defense, cellular metabolism, NCC development or inflammation in cNCC and tNCC from rat embryos exposed to ethanol in vitro. We specifically evaluated expression differences between cNCC and tNCC genes, possibly reflecting the different teratological susceptibilities of the two cell populations.
  
  
  METHODS
  neural tube explants from rat embryos were divided in cranial and trunk portions and used for NCC isolation in vitro on gestational day 10. The migrating cells from the cranial or trunk explants of the neural tube were subsequently exposed to 0 or 88 mmol/l ethanol concentration with or without addition of 0.5 mM N-acetylcysteine (NAC) for 48 h, harvested, and prepared for gene expression measurement by RT-PCR or immunostaining with either distal-less (DLX) or AP 2-alpha antibodies.
  
  
  RESULTS
  evaluation of the immunostained slides showed that approximately 75% of the cNCC and tNCC preparations were of neural crest origin. Exposure to 88 mM ethanol increased the Bax/Bcl-2 ratio in the NCC, and NAC addition diminished this increase. Both cNCC and tNCC upregulated MnSOD and Gpx-1 in response to ethanol, whereas tNCC increased CuZnSOD and EC-SOD after ethanol exposure (cNCC unchanged). Expression of glyceraldehyde-3-phosphate dehydrogenase was downregulated by ethanol in cNCC only. In addition, ethanol exposure caused increased mRNA levels of Pax-3, p53, Vegf-A and decreased expression of Pax-6, Nfe2 in both cNCC and tNCC. Ethanol increased Shh and Bmp-4 and decreased Parp only in cNCC (tNCC unchanged), whereas ethanol exposure increased T box-2 and decreased Gdnf and Ret only in tNCC (cNCC unchanged). In addition, ethanol exposure almost abolished expression of Hox a(1), a(4) and a(5), and left Hox a(2) unchanged in cNCC, whereas all four of these Hox genes were upregulated in tNCC.
  
  
  CONCLUSIONS
  ethanol causes a shift towards apoptosis in both cNCC and tNCC, a shift, which is diminished by NAC treatment. Oxidative defense genes, and genes involved in neural crest cell development are affected differently in cNCC compared to tNCC upon ethanol exposure. Moreover, ethanol downregulates cNCC Hox genes, whereas tNCC Hox genes are upregulated. These patterns of ethanol-altered gene expression may be of etiological importance for NCC-associated maldevelopment in ethanol-exposed pregnancy.
authors.affiliation: Department of Medical Cell Biology, Biomedical Center, PO Box 571, SE-75123, Uppsala University, Uppsala, Sweden. parri.wentzel@mcb.uu.se
authors.forename: Parri
authors.initials: P
authors.lastname: Wentzel
authors.forename: Ulf J
authors.initials: UJ
authors.lastname: Eriksson
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002465:Cell Movement; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D000431:Ethanol / Q000494:pharmacology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007249:Inflammation / Q000235:genetics; D008660:Metabolism / Q000235:genetics; D009432:Neural Crest / Q000187:drug effects* / Q000196:embryology / Q000378:metabolism; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D002492:Central Nervous System Depressants; D016166:Free Radical Scavengers; D012333:RNA, Messenger; D000431:Ethanol; D000111:Acetylcysteine