zyxwv zyxwvutsrq zyxwvutsrqpo zyxwvutsrq zyxwvutsrqponm zyxwvu zyxwvutsrqp OK. ALCOHOLIC LIVER DISEASE, NAFLD AND DRUGINDUCED LIVER DISEASE hepatocyte-specific null mutation of Pten in mice (Pten KO mice) and established these mice as a model of human NASH (JCT I 13; 1774-1 783, 2004). Using this model, our aim is to investigate the effect of N-acetylcystein (NAC), that is widely known to be an anti oxygen agent. Method: In uiuo experiment: Pten KO mice were given NAC (1 mg/ml) in drinking water. Then serum ALT and free radical levels were compared between with and without NAC group. Moreover, histological findings and genes expression of glutathione metabolizer such as glutathione peroxidase (Gpx), glutathione reductase (Gsr) were investigated in livers. In uitro experiment: Overnight cultured Pten KO hepatocytes were preincubated for 2h with NAC (5mM) then washed out, and cells were exposed to 1 0 0 ~ M tert-butyl hydroperoxide (TBuOOH). Cell viability and intracellular level of ROS were measured. The mitochondrial injury of hepatocytes such as mitochondrial permeability transition (MPT) and mitochondrial membrane potential were observed by confocal microscopy. Results: In oioo experinzent: Serum ALT level was significantly lower in NAC-given group at 40 weeks of age. Histological findings indicated that fibrosis and inflammatory cell infiltration were reduced in NAC-given group. Serum free radical level were significantly lower and the genes expression ratio of Gpx to Gsr were significantly increased in NAC-given group. In uitvo experiment: Preincubation of NAC significantly improved cell viability and decreased intracellular ROS levels after exposure to TBuOOH. The mitochondrial depolarization and MPT of Pten KO hepatocytes were supperessed after addition of NAC. Conclusion: Both in vivo and in vitro NAC blocks progression of NASH found in Pten KO mice by reducing ROS. To consider the results of gene expression of glutathione metabolizer, NAC worked by increasing intracellular glutathione. NAC may be a potent therapeutic reagent of human NASH. S281 GI-C: 24.1f27.8 (p=O.OOI), G2-C: 74.2+54.5 (p=O.OOI), GI-G2: 0.32f0.35 (p=0.047), G3-C: p > 0.05, G4-C: 53.2+40.2 (p = 0.001), G3-G4: p >0.05, G5-C: p >0.05, G6-C: 128.8+89 (p=O.OOI), G5-G6: p > 0.05. G2-Exl: p > 0.05, G2-Ex2: 1.98+ I .34 (p > 0.05), G2-Ex3: 4.861t3.03 ( p = 0.01). NFKB expressions were similar across the groups. Conclusion: Ethanol and or LPS suppress the liver mRNA expression of PPARa while same treatments increase TNFa levels. Reversal of PPARa suppression with anti-TNFa treatment in a dose dependent fashion suggests that increased TNFa may be involved in ethanol and/or LPSinduced PPARa suppression. These results suggest that suppression of TNFa may have an implication in the treatment of ethanol induced fatty liver disease by amelioration of altered lipid metabolism. 17481 ROLE OF THE P66SHCA IN ETHANOL-INDUCED OXIDATIVE STRESS AND LIVER DAMAGE zyxwvutsrqpo 17471 TNF-ALPHA MEDIATES THE ETHANOL INDUCED PPAR-ALPHA SUPRESSION IN THE LIVER F.O. Onder’, S. Sengu15, R. Calayoglu5, B. Savas3, K. F. Alcbiyik6, C. Yurdaydin1,2, 0. Uzunalimoglu*, H. Ataoglu4, H. Bozkaya’ . ‘Gastvoentevology Departnzent; Hepatology Institute; ’Pathology Department; 4Molec~larBiology Department, Ankara linioevsiiy Faculty of Medicine, Ankara; 5Nephvology [init, Internal Medicine Department, Ankara University Faculty of Medicine, Ankara; ‘Biochenzistry Department, Hacettepe linioevsiiy Faculty of Medicine, Ankara, Turkey E-mail: oguz.onder@jgmail.com The mechanism of PPARa suppression by ethanol is not understood. In this study, we aimed to test the hypothesis that ethanol-induced PPAR suppression is mediated by TNFa. Method: 96 female Sprague-Downey rats were first divided into 4 groups: ethanol (E), lipopolysaccharide (LPS), E+LPS and control (C) groups and each group was further divided into 2 on the basis of presenceiabsence of anti-TNFa (AT) treatment: Group 1 (n = 16) E+LPS+AT, Group 2: (n = 16): E+LPS, Group 3 (n = 8): LPS+AT, Group 4 ( n = 8): LPS, Group 5 (n = 8): E+AT, Group 6 (n=8): E, Group 7 (n=16): AT, Group C (n=16): C. AT (5 mg/kg) was administered via intraperitoneal route (IP) 12 before the first dose ofethanol. Ethanol (%40 v/v, 2x2.55mg/kg/qd) was administered by gavages for 72 hours and LPS (4 mgikg) was administered by 1P route with the last dose of ethanol. To evaluate the dose response c u v e of PPAR expression, additional 30 rats receiving E+LPS were treated with different doses of AT (Ex 1 I mgikg, Ex2 3 mgilcg, Ex3 10 mg/kg). Serum ethanol (spectrophotometry), TNFa (ELTSA) levels, PPARa and NFKB (Real-Time PCR) expressions in liver tissue were determined. Results: Serum ethanol levels were significantly elevated in the ethanol receiving groups (GI, G2, G5 and G6) when compared to others (p < 0.001). AT treatment suppressed the ethanol and/or LPS-induced TNF elevation (p i 0.001). PPARa mRNA expression was suppressed by ethanol and/or LPS treatments and this suppression was relieved by AT treatment in a dose dependent manner: relative expressions (RE) of PPARa mRNA: S. Fuscol , O.R. Koch3, C. Capone’, S. Borrellol , P. Palozza’ , L.M. Larocca2, A.A. Cravero3, S.M. far^'^, T. Galeottil, G. Panil. ‘Institute of‘ General Pathology; 2Depavtnzent of Pathology, Catholic linioevsiiy Medical School, Ronze, Italy; ‘Department of‘ Pathology, linioevsiiy of’ Bnenos Aires Faculty of Medicine, Bnenos Aives, Argentina E-mail: gpani@rm.unicatt.it Background and Aims: Mice lacking the 66 kD isoform of the adapter molecule ShcA (p66shcA) display increased resistance to oxidative stress and delayed ageing; accordingly, in cultured cell lines, p66 promotes formation of Reactive Oxygen Species (ROS)2 in mitochondria and apoptotic cell death in response to pro-oxidant stimuli. Since mitochondrial ROS and oxidative cell damage are involved in alcohol-induced pathology, we hypothesised that p66 may also have a role in ethanol citotoxicity. Methods: To address the possible role of p66shc in alcohol-induced liver pathology, p66 Knock-out mice and the corresponding p66shc +/+ controls were fed ethanol in the drinking water or a colorie-matched diet witthout ethanol for six weeks. After sacrifice, histological and biochemical changes in liver tissue were evaluated. As an in vitro correlate of ethanol feeding in vivo, p66shc- and mock-transfected 3T3L.1 cells were exposed to alcohol in vitro, and cell response in terms of viability, redox status and protein phosphorylation analysed. Results: In vivo, changes observed in Wild Type mice after 6-week exposure to ethnol in the drinking water, including elevated serum alanine aminotransferase (ALT), liver swelling and evident liver steatosis, were significantly attenuated in p66 -1- mutant mice. p66 protein was significantly upregulated by ethanol in wild type mice, while p66-deficient livers responded to alcohol with a significant upregulation of the mitochondrial antioxidant enzyme MnSOD, nearly absent in control animals. An inverse correlation between expression level of p66 and protection from alcohol-induced oxidative stress and citotoxicity was also confirmed in vitro in 3T3L.1 cells engineered to over-express human p66. In the control cell line, exposure to ethanol elicited the inhibitory (Thr 32) phosphorylation of the Forlchead family transcription factor FKHRL I and the induction of MnSOD, two responses which were constitutively activated and repressed, respectively, in p66-transduced cells. Moreover, p66 increased alcohol-induced ROS and sensitized 3T3-L I cells to ethanol toxicity. Conclusions: Taken together, the above observations clearly indicate a role for p66 in alcohol-induced cell damage, likely via a cell-autonomous mechanism involving impaired expression of antioxidant defenses and mitochondrial distinction. Supported by: Minister0 Affari Esteri, Italian-Argentinian collaborative Research Program (L.40111990) and University of Buenos Aires TM-35.