The liver plays a central role in ethanol metabolism, and oxidative stress is implicated in alcohol-mediated liver injury. β-Catenin regulates hepatic metabolic zonation and adaptive response to oxidative stress. We hypothesized that β-catenin regulates the hepatic response to ethanol ingestion. Female liver-specific β-catenin knockout (KO) mice and wild-type (WT) littermates were fed the Lieber-Decarli liquid diet (5% ethanol) in a pairwise fashion. Liver histology, biochemistry, and gene-expression studies were performed. Plasma alcohol and ammonia levels were measured using standard assays. Ethanol-fed (EtOH) KO mice exhibited systemic toxicity and early mortality. KO mice exhibited severe macrovesicular steatosis and 5 to 6-fold higher serum alanine aminotransferase and aspartate aminotransferase levels. KO mice had a modest increase in hepatic oxidative stress, lower expression of mitochondrial superoxide dismutase (SOD2), and lower citrate synthase activity, the first step in the tricarboxylic acid cycle. N -Acetylcysteine did not prevent ethanol-induced mortality in KO mice. In WT livers, β-catenin was found to coprecipitate with forkhead box O3, the upstream regulator of SOD2. Hepatic alcohol dehydrogenase and aldehyde dehydrogenase activities and expression were lower in KO mice. Hepatic cytochrome P450 2E1 protein levels were up-regulated in EtOH WT mice, but were nearly undetectable in KO mice. These changes in ethanol-metabolizing enzymes were associated with 30-fold higher blood alcohol levels in KO mice. Conclusion : β-Catenin is essential for hepatic ethanol metabolism and plays a protective role in alcohol-mediated liver steatosis. Our results strongly suggest that integration of these functions by β-catenin is critical for adaptation to ethanol ingestion in vivo .

This article discusses several subjects pertinent to a consideration of the role of gender and hormones in alcoholic liver injury (ALI). Beginning with an overview of factors involved in the pathogenesis of ALI, we review changes in sex hormone metabolism resulting from alcohol ingestion, summarize research that points to estrogen as a cofactor in ALI, consider evidence that gut injury is linked to liver injury in the setting of alcohol, and briefly review the limited evidence regarding sex hormones and gut barrier function. In both women and female animals, most studies reveal a propensity toward greater alcohol-induced liver injury due to female gender, although exact hormonal influences are not yet understood. Thus, women and their physicians should be alert to the dangers of excess alcohol consumption and the increased potential for liver injury in females.

OBJECTIVES: Patients with severe acute pancreatitis (AP) typically develop vascular leak syndrome, resulting in hemoconcentration, hypotension, pulmonary edema, and renal insufficiency. Angiopoietin-1 (Ang-1) and 2 (Ang-2) are autocrine peptides that reduce or increase endothelial permeability, respectively. The aim of this study was to determine whether Ang-1 and/or Ang-2 levels are predictive biomarkers of persistent organ failure (>48h) and prolonged hospital course.METHODS: Banked serum from 28 patients enrolled in the Severity of Acute Pancreatitis Study at the University of Pittsburgh Medical Center (UPMC) and 58 controls was analyzed for Ang-1 and Ang-2 levels. Separately, serum from 123 patients and 103 controls at Greifswald University (GU), Germany was analyzed for Ang-2 levels. Angiopoietin levels were measured by enzyme-linked immunosorbent assay.RESULTS: In all, 6 out of 28 UPMC patients (21%) and 14 out of 123 GU patients (13%) developed persistent organ failure and were classified as severe AP. Ang-2 was significantly higher on admission in patients who developed persistent organ failure compared with those who did not in UPMC (3,698 pg/ml vs. 1,001 pg/ml; P=0.001) and GU (4,945 pg/ml vs. 2,631 pg/ml; P=0.0004) cohorts. After data scaling, admission Ang-2 levels showed a receiver-operator curve of 0.81, sensitivity 90%, and specificity 67% in predicting persistent organ failure. In addition, Ang-2 levels remained significantly higher in severe AP compared with mild AP patients until day 7 (days 2-4: P<0.005; day 7: P<0.02). Ang-1 levels were not significantly different between mild and severe AP patients on admission.CONCLUSIONS: Elevated serum Ang-2 levels on admission are associated with and may be a useful biomarker of predicting persistent organ failure and ongoing endothelial cell activation in AP.

Alcoholic liver disease remains a frequent and serious problem for increasing numbers of patients. Research has expanded our molecular understanding of the cellular basis of disease progression; however, translation into therapy is still hampered by a lack of suitable animal models for alcoholic liver disease, as well as from consequences of related liver damage due to malnutrition, hepatitis C virus infection, or abuse of other substances. Many patients with liver disease do not simply consume too much alcohol; they also suffer from comorbidities such as obesity or viral hepatitis, and/or may be addicted to other drugs besides alcohol. This review will summarize the currently available animal models to study liver disease due to either single causes or combinations of liver toxic substances/infections and alcohol.

BACKGROUND The intragastric (IG) ethanol infusion model results in fatty liver, necrosis, inflammation and fibrosis. This model was utilized to study the pathogenesis of alcoholic liver disease (ALD). Disadvantages of the IG model include maintenance of the animals and equipment expense. To develop a voluntary feeding model for ALD, we took advantage of two important observations in the IG model: (i) female rats demonstrate greater severity of alcohol-induced liver injury than males and (ii) rats fed fish oil as a source of fatty acids develop more severe alcoholic liver injury than rats fed other fatty acids with ethanol. METHODS Female Wistar rats (205 to 220 g) were fed for 8 weeks a diet containing 8% ethanol, fish oil (30% of calories), protein, and dextrose. Pair-fed controls (FD) received dextrose in amounts isocaloric to ethanol. The following measurements were made: liver pathology [fatty liver (0 to 4), necrosis, inflammation and fibrosis by Sirius Red], endotoxin and alanine aminotransferase (ALT) in plasma, urine ethanol, lipid peroxidation, nuclear factor kappa-B (NF-kappaB) and mRNA levels for tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Protein levels for iNOS and nitrotyrosine were evaluated by immunohistochemistry and Western Blot analysis. Liver proteasome and cytochrome P450 2E1 activity and protein levels of asialoglycoprotein receptor (ASGPR) were also evaluated. In addition, mRNA levels of fibrogenic markers were assessed. RESULTS All animals lost weight for the initial 2 to 3 weeks but then gained weight until killing at 8 weeks. There was, however, a significant difference (p < 0.05) in weight between the ethanol-fed (Etoh) and (FD) groups at the end of the experiment. The mean urine ethanol levels ranged between 190 and 240 mg/dl. The severity of pathological changes was greater (p < 0.01) in Etoh vs. FD: fatty liver, 3.0 +/- 1.2 vs. 1.2 +/- 0.4; necrosis (foci/mm(2)), 3.9 +/- 2.3 vs. 0.4 +/- 0.3; inflammation (cells/mm(2)), 19.0 +/- 6.3 vs. 1.8 +/- 0.6. Centrilobular collagen deposition (% area), assessed by Sirius Red staining, was greater in Etoh vs. FD. Levels of endotoxin, ALT, CYP2E1 and lipid peroxidation markers were also higher (p < 0.01) in Etoh vs. FD. Levels of NF-kappaB and mRNA of pro-inflammatory mediators (TNF-alpha, COX-2, iNOS) and procollagen-I were increased (p < 0.05) in ethanol-fed rats. Immunohistochemical analysis showed more intense staining for both iNOS and nitrotyrosine in the centrilobular areas in the Etoh vs. FD groups. The greater area of positive staining for iNOS and nitrotyrosine in Etoh vs. FD was confirmed by Western Blot analysis. An increase in the expression of mRNA for profibrogenic genes (p < 0.05) was seen in ethanol-fed rats. CONCLUSIONS A voluntary feeding regimen consisting of fish oil and ethanol in female rats is technically less demanding yet produces pathological and biochemical changes similar to those observed with the IG model. Pathological changes include fatty liver, necrosis and inflammation. Increased NF-kappaB and mRNA and protein levels of the pro-inflammatory mediators TNF-alpha, COX-2 and iNOS, coincided with the presence of necroinflammatory changes. The voluntary feeding regimen is proposed as an alternative to the IG model in the study of alcoholic liver injury.

3663 In recent years, use of complementary and alternative medicine has increased by both healthy and cancer patients. Our previous work showed that many commonly used botanicals demonstrate estrogenic activity in vitro and in vivo. In this study, we examined three botanical extracts that have been reported to inhibit growth of human prostate carcinoma in vitro and in vivo. These extracts are: red clover (Trifolium pretense, RC), used for menopausal symptoms, hormone replacement, cancer prevention, and for digestive and respiratory diseases; milk thistle (Silybum marianum, MT), used as a liver tonic during chemotherapy and for liver disorders and injury; and saw palmetto (Serenoa repens or Sabal serrulata, SP): used for benign prostatic hyperplasia and urinary symptoms. We tested extracts of RC, MT and SP for estrogenicity in vitro using two competitive estrogen receptor (ER) binding assays, with female rat liver as source of ERα and rat prostate for ERβ. Extracts were also tested in the presence and absence of a physiological level of estradiol (E2, 1nM) in ER-positive breast cancer cell lines (MCF-7, predominantly ERα; T47D, predominantly ERβ) and ER-negative (BT-20), and in vivo using ovariectomized (OVX) female rats. In both receptor assays, dose-dependent inhibition of E2 binding was noted with RC and SP. MT competed with E2 at the lowest doses but enhanced binding at highest doses in both assays. In cell proliferation assays, RC caused growth in MCF-7 cells above E2 control levels, whereas in T47D cells, RC had a strong proliferative effect alone and a dramatic additive effect in the presence of E2. In MCF-7, SP was modestly proliferative at the weakest doses yet below E2-stimulated levels, and showed an additive effect with E2. In contrast, in T47D, SP produced a biphasic response, and when combined with E2, a strong additive effect. MT had a proliferative effect in MCF-7 equivalent to E2 stimulation, with an additive effect when combined with E2. In T47D, MT produced modest proliferation, and in the presence of E2, an additive effect beyond E2 stimulation. When fed to OVX female rats at a dose equivalent to 30% of the recommended human dose for 30 days, RC and MT did not increase uterine weight, and SP significantly decreased uterine weight. Serum LH levels were not reduced significantly by RC or MT, but reduced somewhat by SP. Serum E2 levels were not altered but for a slight increase with SP. Hepatic estrogen 2-hydroxylase activity was significantly reduced only by MT. In conclusion, these botanicals demonstrate some degree of estrogenicity. They interact with both ERα and ERβ, and in the presence of a physiological E2 level, may amplify ER-mediated cellular events. Both cell lines demonstrate proliferation in the presence of these extracts, with T47D showing a greater response. While these botanicals may have clinical applications, they should be avoided or used cautiously by patients with hormone-responsive diseases.

AimsThe LAPSG is a multidisciplinary group dedicated to the study of the biology, pathophysiology and disease of the endocrine and exocrine pancreas. In particular the fields of gastroenterology, endocrinology, oncology, surgery, pharmacology, cell and molecular biology, nutrition, immunology and epidemiology will receive attention.It is also the study group’s aim to enhance the communication among scientists from different areas (especially the fields of gastroenterology and endocrinology) who otherwise rarely meet. The means to achieve these goals will be meetings of the members, postgraduate courses, clinical-research workshops and symposia. Pancreatologists and those working in related areas that live and work in Latin America, and who have published at least once during the last 5 years, are warmly invited to join the LAPSG. Members of the LAPSG will automatically become members of the …

Chen RF, Li ZH, Lou SQ and Chen _] S (luargqzlatvu, Cr'1:'m1 Effects of antisense oligonucleotides of PKC-(1 on proliferation and apoptosis of I-lepG, _, in vitro 75 7.1111 l-SH, Yao ZX, I. uo S], _] ia11g IM, Xiao] W, Liu SC, Liu JB, Sun _] M and Pei'. 7, Y CI1ungq:'n_q_ China

Background: Chronic alcohol consumption increases the risk of pancreatitis in humans. Functional hyperstimulation/hypersecretion of the pancreas during chronic alcohol consumption appears to precede the onset of pancreatitis, and may contribute to the increased susceptibility to pancreatitis in alcoholics. However, the origin, nature and timing of hyperstimulation/hypersecretion are unknown. Methods: Male Wistar rats were pair-fed ethanol liquid diet for 15–18 days (including one 9-day dose ramp-up phase) or regular liquid diets before placement of pancreatic, biliary, duodenal and venous catheters. Basal and stimulated pancreatic secretions were measured with or without acute alcohol infusion. Pancreatic secretion was stimulated with intravenous bethanechol, 2-deoxy-D-glucose (2-DG), cholecystokinin (CCK), octapeptide (CCK-8), intraduodenal meal, or vehicle. Results: Acute alcohol potentiated 2-DG stimulated pancreatic secretion (184%, p < 0.05), whereas the response to CCK was unchanged, and the response to bethanechol was decreased (78%, p < 0.05). Short-term alcohol exposure lessened the exaggerated protein secretory response to 2-DG seen in acute alcohol exposure rats and increased the protein response to bethanechol (141%, p < 0.05), CCK (187%, p < 0.05) and meal (217%, p < 0.05). Conclusion: The pancreas is sensitive to acute alcohol ingestion with inhibition of acinar cell function. Rapid adaptation occurs with short-term alcohol feeding, resulting in an exaggerated response to cholinergic input at the acinar cells, plus disinhibition of CCK and meal-stimulated pancreatic secretion. The central response to 2-DG and CCK are similar to area postrema lesions. Adaptation appears to be in response to alcohol-associated inhibition of the neurohormonal stimulatory pathway and compensatory upregulation at the acinar cell level.

Alcohol consumption is a risk factor for chronic pancreatitis (CP), but the mechanism in humans remains obscure because prolonged alcohol consumption in most humans and animal models fails to produce alcoholic chronic pancreatitis (ACP). We hypothesize that the process leading to ACP is triggered by a sentinel acute pancreatitis (AP) event; this event causes recruitment of inflammatory cells, which initiates fibrosis driven by the anti-inflammatory response to recurrent AP and/or chronic oxidative stress. The aim was to determine whether chronic alcohol consumption accelerates fibrosis in response to cerulein-induced pancreatitis in the rat. Wistar male rats were pair-fed control (C) or 5% ethanol (E) Lieber-DeCarli liquid diets. Animals were studied without pancreatitis (P0), with cerulein pancreatitis induced once (P1), or with cerulein-induced pancreatitis weekly for 3 weeks (P3). AP markers, inflammation, and fibrosis were measured histologically, by gene expression profiling and protein expression. Macrophage infiltration was reduced in EP0 versus CP0 rats, but the pattern was reversed after AP. Microabscess, severe necrosis, and early calcification were only induced in the EP3 rats. Fibrosis was significantly induced in the EP3 rats versus EP1, CP1, and CP3 by histology, hydroxyproline content, and mRNA expression for collagen alpha1(1) and procollagen alpha2(1). Proinflammatory cytokine mRNAs were up-regulated shortly after induction of AP, while the anti-inflammatory cytokines (interleukin-10 and transforming growth factor-beta) were strongly up-regulated later and in parallel with fibrogenesis, especially in the EP3 rats. Pancreatic fibrosis develops after repeated episodes of AP and is potentiated by alcohol. Expression of fibrosis-associated genes was associated with expression of anti-inflammatory cytokines in alcohol-fed rats.

5786 Several traditional Chinese medicines (TCM) are used to stimulate the immune system, in order to maintain health and as an adjuvant to chemotherapy. Previous work has shown that certain medicinal botanicals traditionally used for gynecological complaints have estrogenic properties, likely because of phytoestrogen content. The aims of this study were to examine if certain TCM herbs altered proliferation in breast cancer cells that are estrogen receptor (ER)-positive but differ in the expression of ERα and ERβ, and if these herbs altered cellular response to estradiol (E2). The cell lines MCF-7 (60:40 ERα:ERβ) and T47D (33:67 ERα:ERβ) were treated with various dilutions of standard extracts of dang gui (DG, angelica), ling zhi (LZ, reishi mushroom), gang cao (GC, licorice), he show wu (HSW, fo-ti), huang qi (HQ, astragalus) in the presence or absence of E2 (1nm). Controls were E2 treatment only and no treatment. After 72 hours, proliferation index was determined via a standard assay; values for herbal treatments were compared to those of controls. The two cell lines differed in response to E2, in that MCF-7 responded more strongly to E2 than did T47D (1.99 vs. 1.70). The most noteworthy difference between these two cell lines was seen in their responses to HSW, LZ, and DG. MCF-7 either had no response (HSW) or a slight response (DG and LZ) to these herbal extracts. T47D, on the other hand, exhibited a dose-related increase in proliferation at higher doses of HSW, LZ and DG, with DG and LZ exceeding the E2 control levels. This differential response was also seen when both cell lines were treated with these herbs along with E2. In MCF-7, E2 tended to mask the effects, if any, of these herbs. In contrast, in T47D cells, addition of E2 to both LZ and DG produced an additive effect, with proliferation index exceeding E2 levels at the higher doses. HSW with E2 in T47D cells had no effect on E2-induced proliferation. HQ alone was biphasic in both cell lines, with proliferative effects at highest doses reaching E2 control levels in MCF-7 and exceeding E2 control values in T47D. HQ with E2 had no effect on the E2-induced proliferation in MCF-7; however, in T47D cells, it maintained a biphasic response with enhancement of proliferation at higher doses. GC alone exhibited a proliferative effect equivalent to that of the E2 control in MCF-7; in T47D, GC produced a strong dose response exceeding that of the E2 control. These responses did not change with the addition of E2 to GC for both cell lines.These results indicate that TCM herbs used for immune system support may be estrogenic, and that the response differs depending on the cell line and the presence or absence of E2. In general, T47D cells are better responders to these herbs, most likely because of their higher content of ERβ, which has a higher affinity than ERα for phytoestrogens. The estrogenicity of these herbal preparations should be taken into account by those who want to reduce their estrogen exposure.

Chronic alcohol ingestion appears to increase susceptibility of the pancreas to pancreatitis through multiple mechanisms. The aim of the current study was to determine the effect of chronic low- and high-dose alcohol consumption on the neurohormonal control of the exocrine pancreas in rats. Male Wistar rats were fed Lieber DeCarli liquid control-, low-, and high-dose alcohol diets for 3 months. Pancreatic exocrine secretion was measured under basal and 2-deoxy-D-glucose (2-DG)-, CCK-, bethanechol-, or meal-stimulated conditions while on chronic alcohol diets and after 2-DG or CCK stimulation during alcohol withdrawal in awake rats. Chronic alcohol ingestion was associated with a dose-related inhibition of basal pancreatic protein secretion, which was reversed upon alcohol withdrawal. Low-dose alcohol feeding had no effect on bethanechol-stimulated pancreatic secretion but altered 2-DG-stimulated pancreatic secretion. In chronic high-dose alcohol rats, meal- and bethanechol-stimulated protein secretion was significantly potentiated during early and late phases. The response to CCK appeared to be disinhibited, whereas the response to 2-DG was uniformly blunted. Upon withdrawal of low-dose alcohol, the response to 2-DG was potentiated, whereas with the withdrawal of high-dose alcohol, the response to CCK was potentiated. Adaptation to chronic alcohol consumption differs depending on the alcohol dose. The most significant effects were seen after high-dose alcohol withdrawal, with apparent loss of central inhibitory regulation combined with exaggerated response at the acinar cell level. This combination of factors could increase susceptibility to acute alcoholic pancreatitis through a hyperstimulation mechanism.