patients, linear modeling was done using the R package limma to identify differentially-expressed genes in livers with advanced fibrosis (n=19) versus early fibrosis (n=20). Signific-ance was set at a False Discovery Rate of 5% after Benjamini-Hochberg correction. TheWilcoxon rank-sum test with multiple testing threshold q<.05 was then used to compareexpression in exposed and unexposed patients with advanced fibrosis in order to assessantagonism or potentiation by alcohol exposure. Permutation analysis was also performedto confirm the findings. Results: In the 39 non-drinking (unexposed) patients, 585 geneswere found to be differentially expressed in patients with advanced fibrosis vs. early fibrosis,identifying a group of fibrosis-associated genes. In the 13 alcohol-exposed patients withadvanced fibrosis, expression of only one of these genes (COL16A1) was potentiated, whereasthe fibrosis-associated pattern of 338 other genes (including genes associated with extracellu-lar matrix (COL1A2, FBN1); PDGF signaling (STAT1); oxidoreductases (ALDH1A3,KDM5A); integrin cell surface interactions (LAMA2); and vascular development(CXCL12,VEGFC, TGFB2) was antagonized. Histology demonstrated that the alcohol-exposed patients were more likely to have early fibrosis than unexposed patients (62.5%vs. 51.2%; p=.002). They were also younger (47±2 vs. 53±2 years; p=0.015) and morelikely to be male (51.5% vs. 20.5%; p=.002). Race, BMI, diabetes mellitus, hypertension,and triglyceride levels did not differ significantly by exposure. Conclusions: In this cohortof NAFLD patients, alcohol exposure was associated with less advanced fibrosis and generallyattenuated the changes in liver gene expression that accompanied fibrosis progression innon-drinkers.

986

Female Mice Are More Susceptible to Ethanol-Induced MitochondrialDepolarization: Studies by Intravital Multiphoton MicroscopyQinlong Liu, Hasibur Rehman, Venkat K. Ramshesh, Yanjun Shi, John J. Lemasters, ZhiZhong

Background: Mitochondrial dysfunction may contribute to the pathogenesis of alcoholicliver disease (ALD). Our previous study showed that acute ethanol treatment causes awidespread but reversible mitochondrial depolarization In Vivo. It is well known that womenhave higher risk of developing ALD. It remains unknown whether mitochondrial dysfunctioncontributes to this increased susceptibility of females to ethanol-induced liver injury. Aim:This study compared mitochondrial polarization status in living male and female mice inrelation to other pathogenic effects after acute ethanol treatment. Methods: Male and femalemice of the same age were gavaged with one dose of ethanol (1 - 6 g/kg). Mitochondrialpolarization, fat accumulation and cell death were visualized by intravital multiphotonmicroscopy of rhodamine 123 (Rh123), BODIPY493/503 and propidium iodide (PI), respect-ively. Results: In male mice, mitochondrial depolarization was rare after saline treatmentbut occurred as early as 1 h after acute ethanol treatment (6 g/kg, ig). After 6 h, ~92% ofhepatocytes contained depolarized mitochondria. Subsequently, mitochondria in 84% ofhepatocytes repolarized at 24 h. Ethanol-induced mitochondrial depolarization occurred ina dose-dependent manner in a range of 1 - 6 g/kg. In female mice gavaged with saline,mitochondrial depolarization was observed in ~2.5% of hepatocytes, which was slightlyhigher than in male mice. When mice were given ethanol of 1 - 4 g/kg, mitochondrialdepolarization was higher in females than males at all doses. The ratios of mitochondrialdepolarization between females and males were ~2.8 fold in 1 g/kg of ethanol and ~1.5 foldin 4 g/kg of ethanol. Mitochondrial depolarization occurred in ~90% hepatocytes whenfemale mice were given ethanol of 4 g/kg. Increased mitochondrial depolarization in femaleswas associated with lower hepatic ATP levels and more severe hepatic steatosis as detectedby BODIPY493/503 fluorescence in living mice and by Oil-Red-O staining in liver slides.Serum ALT levels and hepatocyte apoptosis were also slightly higher in females than inmales. At 2 weeks after ovariectomy, mitochondrial depolarization was blunted by ~57%in female mice after ethanol treatment. Conclusion: Acute ethanol causes widespread,reversible hepatic mitochondrial depolarization in livingmice. Female mice are more suscept-ible to mitochondrial dysfunction than male mice, which may contribute to occurrence ofethanol-induced liver injury. Female hormones most likely play an important role in thishigher susceptibility to mitochondrial toxicity of ethanol (NIAAA).

987

Prevalence and Risk Factors for Alcohol Recidivism Among LiverTransplantation Recipients: A Single Center Anonymous SurveyKevin Cronley, Shweta Nagendra, Mary Penko, Gregory S. Cooper, Pierre M. Gholam

Introduction: Alcoholic Liver Disease (ALD) is the second leading indication for liver trans-plantation (LT) in the United States. The prevalence of recidivism after LT and its impacton graft and patient survival remain controversial. The aim of our study was to survey theprevalence of recidivism among patients transplanted for ALD and other etiologies (non-ALD). We also sought to determine risk factors for recidivism among LT recipients. Methods:We sent surveys to 230 closely followed adult LT recipients at our institution. Respondentswere assigned an anonymous study code for data linkage to study parameters which werede-identified. Survey mailing envelopes were labeled with the study code and instructionsnot to include any identifiers. Parameters of interest included duration of abstinence pre-LT, quantity of alcohol consumed daily in standard drinks pre and post-LT, illicit substanceand tobacco use, counseling on abstinence before and after LT and perception of whetheralcohol consumption post-LT was harmful. Respondents were informed that the investigatorswould be blinded to their identity at all times. Results: 92 patients (40%) responded andincluded 26 transplanted for ALD and 66 for non-ALD. Seven (27%) responders transplantedfor ALD had alcohol recidivism post-transplant versus 27 (41%) of those transplanted fornon-ALD (p=0.210, Odds Ratio (OR) 95% Confidence Interval (CI) 0.196-1.440). 92% ofsober responders considered complete abstinence post-transplantation as the only safe optionfor their liver versus 62% of recidivists (p=0.04, 95% CI 0.38-1.18). In addition, 62% ofrecidivists in the ALD group considered their level of alcohol consumption harmful versus66% in the non-ALD group (p=0.41, OR 95% CI 0.43-7.82). 78% of respondents in theALD group reported receiving counseling on abstinence post-LT versus 70% in the non-ALD group (p=0.585, OR 95% CI 0.46-4.66). When all respondents were analyzed, soberpatients post-LT had a significantly lower prevalence of any tobacco consumption versus

S-915 AASLD Abstracts

recidivists, both pre-LT (37% vs. 71%; p<0.001, OR 95% CI 3.85-119.14) and post-LT(11% vs. 71% p=0.034, OR 95% CI 3.32-65.76). There were no statistically significantdifferences in the duration of abstinence pre-LT greater or less than 6 months (p=0.26, OR95% CI 0.37-1.32), the amount of alcohol consumed per day pre-LT (p=0.56, OR 95% CI0.66-2.78) or the use of any or no illicit substance pre-LT (p=0.39, OR 95% CI 0.05-2.33).Conclusions: Among LT recipients, any tobacco use pre and post-LT as well as a perceptionthat alcohol use post-LT is safe appear to be associated with recidivism. These data suggestthat an additional focus on education targeting smokers before and after LT as well asincreasing efforts towards tobacco cessation may result in lower recidivism rates in the LTtransplant recipient.

988

Impaired Protein Trafficking and Elevated Triglycerides in a Mouse Model ofEthanol AdministrationKathryn E. Lazure, Jacy L. Kubik, Karuna Rasineni, Benita McVicker, Carol A. Casey

Background: We have previously identified multiple ethanol-induced impairments to theendocytic function of several protein receptors. Currently we are examining how ethanoladministration affects trafficking of lipid droplets (LDs), which are neutral fat stores sur-rounded by a thin protein coat. We have identified ethanol-induced alterations in severalRab proteins which are involved in both protein and lipid trafficking in a rat model. In thepresent study we examined a time course for ethanol-induced impairments in proteintrafficking and also how ethanol administration might alter hepatic lipid stores in a mousemodel. Methods: Alcohol administration: Wild-type mice (C57Bl/129SV F2 hybrids) werefed the Lieber-DeCarli control or ethanol diet for periods up to 3.5 weeks. Ethanol contentwas incrementally increased by 1% per day up to a final 5% concentration, which wascontinued up to 3 weeks. Surface Binding of ASOR (asialoorosomucoid): Surface bindingof 125-I ASOR on hepatocytes was measured at 4 degrees C. Western Blot (WB) Analysis:Total cellular ASGPR was detected after incubation with anti-ASGP-R antibody. Amountsof Rabs 1,2 and 5 were determined in LDs and total liver fractions using appropriateantibodies. ALT Determination: ALT was assayed in serum obtained at the time of sacrificeusing VITROS Chemistry System (Ortho Clinical Diagnostics). Triglyceride (TG) Assay: TGlevels were determined using Folch Extraction. LD isolation: LDs were isolated by standardcentrifugation techniques involving a discontinuous sucrose gradient. Results: Surface Bind-ing: No impairment in ASOR surface binding was identified after the earliest period (1 dayof 5% ethanol) , however, significant decreases (55-60%) in binding were observed afterone week of 5% ethanol administration, and this impairment continued over the time course.ASGPR total protein content: Alterations in ASGPR receptor protein content were significantlydecreased at 2-3 weeks. Serum ALT level: No elevation of liver enzyme levels was identifieduntil after the 3 week feeding period. Triglyceride Determination: Ethanol fed mice (3 weeks)had a significant increase (3-fold) in triglyceride content (expressed as mg triglyceride perliver) compared to the controls. Rab proteins: The presence of Rabs 1,2 and 5 were identifiedin the LD fractions isolated from the mouse livers. Conclusions: ASGPR surface binding isimpaired and significant triglyceride elevation is noted in mice fed alcohol for 3 weeks. Inaddition, the LD fractions of these livers showed the presence of several Rab proteins, oneof which in particular (Rab 5) is involved in secretory protein trafficking. This mousemodel of ethanol administration will prove very useful for studies of both protein andlipid trafficking.

1061

A Crucial Role of Hepatocyte p53 in Liver FibrogenesisTakahiro Kodama, Tetsuo Takehara, Satoshi Shimizu, Hayato Hikita, Minoru Shigekawa,Hinako Tsunematsu, Takuya Miyagi, Atsushi Hosui, Tomohide Tatsumi, Hisashi Ishida,Tatsuya Kanto, Naoki Hiramatsu, Norio Hayashi

The tumor suppressor p53 primarily functions as a guardian of the genome, suppressingtumor development in various organs. Aside from its well-established role, recent reportshave revealed that p53 is involved even in the pathophysiology of various non-tumorousconditions such as insulin resistance, cardiac failure and early aging. According to the liverdisease, while p53 accumulation has been observed in hepatocytes of chronic liver diseases,the precise role of p53 in liver fibrosis is unclear. To this end, we generated mice withhepatocyte-specific deletion of Mdm2, a critical p53 inhibitor, which strictly maintains p53at a low level by promoting p53 degradation via the ubiquitin/proteasome pathway. Thesemice revealed that hepatocyte p53 activation caused spontaneous liver fibrosis with increasedapoptosis and expression of the connective tissue growth factor (CTGF) gene in hepatocytes.p53 deletion in Mdm2 knockout mice completely abolished these phenotypes. In Vitroexperiments revealed that p53 upregulated CTGF via repression of the miR-17-92 gene inHepatocytes. Human liver samples showed significant correlation between CTGF and p53-regulated gene expression, both of which increased in the fibrotic liver. The present studyunveiled a previously unrecognized role of p53 in inducing CTGF expression and promotingliver fibrosis, thus suggesting that the p53 and CTGF pathway may be a therapeutic targetfor liver fibrosis.

1062

Activated Hepatic Stellate Cells Transcriptionally Upregulate Ecto-5'-Nucleotidase/CD73 via Specific Promoter ElementsMichel Fausther, Jonathan A. Dranoff

Background. Extracellular adenosine is a potent regulator of liver fibrosis and inflammation.Ecto-5'-nucleotidase/CD73 is the rate-limiting enzyme in the generation of extracellularadenosine. Furthermore, mice lacking CD73 are resistant to experimental liver fibrosis andhave impaired adenosine generation within the liver (Peng Z, FASEB J. 2008). However,the cellular expression and regulation of CD73 within the fibrotic liver have not been defined.Aims. To determine the molecular mechanisms regulating CD73 expression in hepaticstellate cells (HSC). Methods. CD73 expression in rat liver sections that had undergone

AA

SL

DA

bst

ract

s