title: S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism.
abstract: OBJECTIVE
  To determine the role of c-Jun N-terminal kinase (JNK) activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosyl-methionine (AdoMet).
  
  
  METHODS
  Primary hepatocyte cultures were pretreated with 100 micromol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays. JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by Ellman's method and reactive oxygen species (ROS) generation by cell cytometry.
  
  
  RESULTS
  The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decrease in ethanol-induced apoptosis (P< 0.05). AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity, and prevented cytochrome c release and pro-caspase 3 cleavage.
  
  
  CONCLUSIONS
  The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease (ALD) treatment.
authors.affiliation: Centro de Investigacion y Estudios Avanzados IPN, Av. IPN No. 2508 Mexico DF, CP 07360, Mexico.
authors.forename: Maria del Pilar
authors.initials: Mdel P
authors.lastname: Cabrales-Romero
authors.forename: Lucrecia
authors.initials: L
authors.lastname: Márquez-Rosado
authors.forename: Samia
authors.initials: S
authors.lastname: Fattel-Fazenda
authors.forename: Cristina
authors.initials: C
authors.lastname: Trejo-Solís
authors.forename: Evelia
authors.initials: E
authors.lastname: Arce-Popoca
authors.forename: Leticia
authors.initials: L
authors.lastname: Alemán-Lazarini
authors.forename: Saúl
authors.initials: S
authors.lastname: Villa-Treviño
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D008297:Male; D008928:Mitochondria; D051381:Rats; D011916:Rats, Inbred F344; D017382:Reactive Oxygen Species / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology
chemical_list: D000873:Anthracenes; D051023:BH3 Interacting Domain Death Agonist Protein; C494816:Bid protein, rat; D017382:Reactive Oxygen Species; C432165:pyrazolanthrone; D000431:Ethanol; D012436:S-Adenosylmethionine; D045304:Cytochromes c; D048031:JNK Mitogen-Activated Protein Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione