title: Ethanol effect on cell proliferation in the human hepatoma HepaRG cell line: relationship with iron metabolism.
abstract: BACKGROUND
  Alcoholism increases the risk of cirrhosis and/or hepatocellular carcinoma development. Iron, like ethanol, modulates the cell growth. However, the relationship between alcohol and iron toward hepatocyte proliferation has not been clearly elucidated. The purpose of this study was to evaluate, in the human HepaRG cell line model, the impact of ethanol on hepatocyte proliferation in relation to modulations of iron metabolism and the protective effect of iron metabolism manipulation by chelators in alcohol liver diseases.
  
  
  METHODS
  The human hepatoma HepaRG cell line model was used. Cell viability was determined by measuring succinate dehydrogenase activity, total protein level by the Bradford method. DNA synthesis was evaluated by [(3)H]-methyl thymidine incorporation. Cytotoxicity was studied by release of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) in culture medium and apoptosis by measuring caspase 3/7 activity. Gene expression was analyzed by RT-qPCR. Total iron, soluble transferrin receptor, and ferritin levels were, respectively, measured by colorimetrical, immuno-nephelometrical, and immuno-turbidimetrical methods. Intracellular iron uptake and accumulation was examined by radionuclide (55)Fe (III) measurement and Perls staining.
  
  
  RESULTS
  Results showed that ethanol decreased all the parameters associated with HepaRG cell proliferation (cell viability, total protein levels, and DNA synthesis) in a dose- and time-dependent manner. This effect was accompanied by cytotoxicity and apoptosis as evaluated by a significant increase in extracellular enzymes (LDH, AST, ALT) and caspase 3/7 activity, respectively. Ethanol exposure was accompanied by an increased cellular iron uptake, together with increased expression of genes involved in iron transport and storage such as l-ferritin, Divalent Metal transporter 1, transferrin, transferrin receptor 1, and ceruloplasmin. Ethanol impact was intensified by iron-citrate and decreased by iron chelators when added to the culture medium.
  
  
  CONCLUSIONS
  The results indicated that (i) ethanol-induced iron metabolism dysfunction could be one of the underlying mechanisms of ethanol antiproliferative effect and (ii) exogenous iron may accentuate ethanol hepatoxicity. These data suggest that iron metabolism manipulation by chelators may be a useful therapeutic approach in alcohol-related liver diseases.
authors.affiliation: Inserm UMR, "Foie, Métabolismes et Cancer," Rennes, France.
authors.forename: Thi Hong
authors.initials: TH
authors.lastname: Tuoi Do
authors.forename: François
authors.initials: F
authors.lastname: Gaboriau
authors.forename: Martine
authors.initials: M
authors.lastname: Ropert
authors.forename: Romain
authors.initials: R
authors.lastname: Moirand
authors.forename: Isabelle
authors.initials: I
authors.lastname: Cannie
authors.forename: Pierre
authors.initials: P
authors.lastname: Brissot
authors.forename: Olivier
authors.initials: O
authors.lastname: Loréal
authors.forename: Gérard
authors.initials: G
authors.lastname: Lescoat
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000633:toxicity*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism*
chemical_list: D000431:Ethanol; D007501:Iron