e28 Abstracts / Digestive and Liver Disease 46 (2014) e18–e42 via dimethylarginine-dimethylaminohydrolase (DDAH). This study investigated whether changes in serum levels of ADMA occur in a rat model of non-alcoholic steatohepatitis (NASH) and what is the mechanism involved. Materials and methods: NASH was induced in Male Wistar rats by 8 weeks of feeding with an MCD diet (methionine/choline- deficient diet). Blood samples and hepatic biopsies were collected after 1, 2, 3, 4 and 8 weeks. Serum hepatic enzymes (AST, ALT and g-GT) and ADMA were evaluated. Hepatic biopsies were used for in situ NAD(P)H autofluorescence detection and for mRNA expres- sion of DDAH and ADMA transporters (CAT-1) by RT-PCR. Tissue DDAH activity and content of lipid peroxides, glutathione and ATP were also quantified. Results: NASH injury was confirmed by altered serum levels of hepatic enzymes. A time dependent decrease in serum ADMA levels and an increase in mRNA expression of DDAH and CAT-1 were found. The hepatic DDAH activity decreased with a concomitant increase in oxidative stress, as demonstrated by high lipid peroxide levels and low GSH content. A decrease in ATP levels and in the NAD(P)Hbound/free ratio reflecting the mitochondria alterations were detected. Conclusions: These results indicate that the oxidative stress observed can contribute to the reduction of DDAH activity. This enzyme is a cysteine hydrolase that may be inhibited by increased reactive oxygen species associated to mitochondria dysfunction. The observed decrease in serum ADMA may be due to the increase in ADMA transporter, CAT-1. These data confirm and support the crucial role of the liver in the control of ADMA levels by taking up large amounts of ADMA from the systemic circulation. (Supported by Fondazione Cariplo, grant no. 2011-0439.) http://dx.doi.org/10.1016/j.dld.2014.01.064 T-25 ENHANCER OF ZESTE HOMOLOG 2 (EZH2) EXPRESSION AND ACTIVATION IN IN VIVO AND IN VITRO NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD) D. Gnani 1 , S. Vella 2 , A. Crudele 1 , S. Ceccarelli 1 , C. De Stefanis 1 , S. Gaspari 2 , V. Nobili 1 , F. Locatelli 2 , V.E. Marquez 3 , R. Rota 2 , A. Alisi 1 1 Liver Research Unit, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy 2 Department of Oncohematology, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy 3 Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, CCR, National Cancer Institute, NIH, Frederick, Maryland Introduction: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease in Western countries. NAFLD devel- opment/progression depends on complex interactions between genetic background, epigenetic changes and environmental factors. The trimethylation of the Lysine 27 on histone H3 (H3K27me3), by the Enhancer of Zeste Homolog 2 (EZH2) protein, is one of the most relevant epigenetic mechanisms. Although, it is well known that EZH2 activity may induce transcriptional repression of genes and microRNAs, there is no evidence of its potential expression/activity during NAFLD pathogenesis. Aim: In the present study we investigated the EZH2 expres- sion/activity in vivo and in vitro NAFLD and its potential correlation with liver damage. Materials and methods: We performed biochemical and molec- ular analyses to evaluate EZH2 expression/activity in the liver from high-fat/high-fructose diet fed (HFa/HFr-D) rats and in free fatty acids (FFAs)-treated HepG2 cells. 3-Deazaneplanocin A (DZNep), a pharmacological inhibitor of EZH2 was used to evaluate its effects on hepatocellular damage. Results: Our results demonstrated that EZH2 protein nuclear expression and its activity on H3K27me3 were down-regulated in livers from HFa/HFr-D fed rats and in FFAs-treated HepG2 cells. Low levels of EZH2 showed an inverse correlation with lipid accu- mulation and with the expression of pro-inflammatory markers, such as TNF-alpha and TGF-beta, and the expression of miR-200b and miR-155 in in vivo and in vitro NAFLD. Moreover, the use of DZNep, further increased: lipid accumulation, TNF-alpha and TGF-beta transcription, and miR-200b and miR-155 levels in FFAs- treated HepG2. Conclusions: In conclusion we demonstrated, for the first time, that NAFLD is characterized by reduction of nuclear EZH2 expres- sion/activity, and the treatment with DZNep may render HepG2 more susceptible to lipid accumulation and inflammation. Further investigations could be useful to clarify EZH2 role in the liver dam- age due to diet-induced NAFLD, as well as to develop potential targeted-preventive strategies. http://dx.doi.org/10.1016/j.dld.2014.01.065 T-26 P53 STATUS AFFECTS COPPER HOMEOSTASIS IN EXPERIMENTAL MODEL OF HEPATIC STEATOSIS M. Arciello 1,2 , A. Longo 1 , M. Gori 1 , B. Barbaro 1 , C. Viscomi 1 , R. Maggio 1 , C. Balsano 1,3 1 Francesco Balsano Foundation, Rome, Italy 2 Department Internal Medicine and Medical Specialties, “Sapienza” University of Rome, Rome, Italy 3 Institute of Molecular Biology and Pathology (IBPM) – CNR, Rome, Italy Nonalcoholic fatty liver disease (NAFLD) is a pathological con- dition, ranging from simple steatosis to steatohepatitis, which can progress up to hepatocarcinoma. P53 protein has been proposed as new player in NAFLD. One of its target is SCO2, a copper (Cu) chaperone essential for maintenance of Cu homeostasis, which it is implicated in the Cu secretory pathway. Cu unbalance affects lipid metabolism and seems involved in NAFLD. Through an in vitro model of steatosis we investigated the possible role of p53 status in Cu homeostasis in NAFLD. HepG2 (wt-p53) and Huh7.5.1 (Y220C mutant p53) cells were treated for 14 and 24 h with Free Fatty Acids (FFAs), oleic and palmitic acids (2:1 ratio, final concentration 0.5 mM). Intracellular lipids and cytotoxic effects were evaluated by AdipoRed and Ala- marBlue assays. Intracellular Cu content was measured by Atomic Absorption Spectrometry. mRNA and protein levels of p53, its target genes and some genes involved in copper trafficking were analysed by qRT-PCR and Immunoelectrophoresis. In both cell lines treatment increased lipid content and was not cytotoxic. The treatment caused a different p53 response in HepG2 and Huh 7.5.1. In fact, after 14 h, p53 was not affected in HepG2, whereas, in Huh 7.5.1 it was up-regulated. At 24 h, instead, we observed an up-regulation of the wt-p53 and a down-regulation of the mutated one. Phosphorylated p53 and its target p21 have the same trend of p53 in both cell lines. In HepG2 FFAs did not affect Cu content; conversely, in Huh 7.5.1 Cu amount was reduced after 24 h. Accordingly, while in HepG2 the early activation of Cu secretory pathway is counterbalanced by p53 activation, in Huh 7.5.1, this modulation did not occur.