title: Mitochondrial dna damage and impaired mitochondrial function contribute to apoptosis of insulin-stimulated ethanol-exposed neuronal cells.
abstract: BACKGROUND
  Ethanol inhibition of insulin signaling may contribute to impaired central nervous system development in fetal alcohol syndrome. An important consequence of ethanol inhibition of insulin signaling is increased apoptosis due to reduced levels of insulin-stimulated phosphoinositol-3-kinase activity.
  
  
  METHODS
  We used viability assays, end-labeling, Western blot analysis, and MitoTracker (Molecular Probes, Eugene, OR) fluorescence labeling to determine whether ethanol-induced central nervous system neuronal cell death was mediated in part by increased mitochondrial (Mt) DNA damage and impaired Mt function.
  
  
  RESULTS
  In ethanol-exposed, insulin-stimulated PNET2 central nervous system-derived human neuronal cells, reduced viability was associated with increased Mt DNA damage, reduced Mt mass (manifested by reduced Mt protein expression and MitoTracker Green fluorescent labeling), and impaired Mt function (manifested by reduced levels of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide activity, cytochrome oxidase-Complex IV, Subunit II expression, and MitoTracker Red fluorescence). The adverse effects of ethanol on Mt function were reduced by pretreating the cells with broad-spectrum caspase inhibitors and nearly abolished by nerve growth factor stimulation, with or without concomitant treatment with global caspase inhibitors.
  
  
  CONCLUSIONS
  These results suggest that ethanol-induced death of insulin-stimulated immature neuronal cells is mediated in part by impaired Mt function associated with Mt DNA damage and reduced Mt mass, and therefore it is likely to contribute to neuronal loss associated with fetal alcohol syndrome. The findings also suggest that the adverse effects of ethanol on insulin-stimulated survival and metabolic function could be overcome by stimulating with growth factors that support Mt function through insulin-independent pathways.
authors: Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island 02903, USA. delamonte@hotmail.com
authors: S M
authors: SM
authors: de La Monte
authors: J R
authors: JR
authors: Wands
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D061945:Caspase Inhibitors; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects; D004272:DNA, Mitochondrial / Q000096:biosynthesis / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology; D009474:Neurons / Q000737:chemistry / Q000187:drug effects* / Q000648:ultrastructure; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis; D011506:Proteins / Q000032:analysis
chemical_list: D061945:Caspase Inhibitors; D004272:DNA, Mitochondrial; D004791:Enzyme Inhibitors; D007328:Insulin; D011506:Proteins; D000431:Ethanol; D011451:Prostaglandin-Endoperoxide Synthases