Obeticholic acid reduces biliary and hepatic matrix metalloproteinases activity in rat hepatic ischemia/reperfusion injury.

BACKGROUND: We have previously shown that obeticholic acid (OCA) upregulates the biliary excretion of asymmetric dimethylarginine (ADMA), an inhibitor of iNOS regulating the activity of matrix metalloproteinases (MMPs). Here, the effects of OCA on MMP-2 and MMP-9 activity in liver, bile and serum were evaluated after hepatic ischemia/reperfusion (I/R) injury. MATERIAL AND METHODS: Male Wistar rats (n = 20) were orally administered 10 mg/kg/day of OCA (5 days) and subjected to a 60-min ischemia and 60-min reperfusion. Bile, serum and tissue were collected for MMP-2 and MMP-9 activity quantification. The MMP regulator tissue reversion-inducing cysteine rich protein with Kazal motifs (RECK), tissue inhibitor of metalloproteinases (TIMPs), iNOS and biliary levels of LDH, γGT, glucose and ADMA were quantified. RESULTS: In the I/R group, OCA administration reduced MMP-2 and MMP-9 in liver, bile and serum. A downregulation of tissue RECK and TIMPs, observed under I/R, were recovered by OCA. Immunohistochemical staining of hepatic tissue demonstrated that RECK expression is mainly localized in both cholangiocytes and hepatocytes. Hepatic iNOS positively correlated with tissue MMP-2 and MMP-9 activity. Biliary levels of LDH, γGT and glucose were lower in I/R rats treated with OCA; in bile, MMP levels positively correlated with LDH and γGT. CONCLUSION: Thus, OCA administration confers protection to cholangiocytes via downregulation of biliary MMPs in livers submitted to I/R. This event is associated with hepatic RECK- and TIMP-mediated MMP decrease.

Efficacy and safety of pemafibrate administration in patients with dyslipidemia: a systematic review and meta-analysis.

BACKGROUND: Using a meta-analysis of randomized controlled trials (RCTs), this study aimed to investigate the efficacy and safety of pemafibrate, a novel selective peroxisome proliferator-activated receptor α modulator, in patients with dyslipidemia. METHODS: A search was performed using the MEDLINE, Cochrane Controlled Trials Registry, and ClinicalTrials.gov databases. We decided to employ RCTs to evaluate the effects of pemafibrate on lipid and glucose metabolism-related parameters in patients with dyslipidemia. For statistical analysis, standardized mean difference (SMD) or odds ratio (OR) and 95% confidence intervals (CIs) were calculated using the random effect model. RESULTS: Our search yielded seven RCTs (with a total of 1623 patients) that satisfied the eligibility criteria of this study; hence, those studies were incorporated into this meta-analysis. The triglyceride concentration significantly decreased in the pemafibrate group (SMD, - 1.38; 95% CI, - 1.63 to - 1.12; P < 0.001) than in the placebo group, with a reduction effect similar to that exhibited by fenofibrate. Compared with the placebo group, the pemafibrate group also showed improvements in high-density and non-high-density lipoprotein cholesterol levels as well as in homeostasis model assessment for insulin resistance. Furthermore, the pemafibrate group showed a significant decrease in hepatobiliary enzyme activity compared with the placebo and fenofibrate groups; and, total adverse events (AEs) were significantly lower in the pemafibrate group than in the fenofibrate group (OR, 0.60; 95% CI, 0.49-0.73; P < 0.001). In contrast, the low-density lipoprotein cholesterol level was significantly higher in the pemafibrate group than in the placebo (P = 0.006) and fenofibrate (P < 0.001) groups. CONCLUSIONS: The lipid profile significantly improved in the pemafibrate group than in the placebo group. In addition to the pemafibrate group having an improved lipid profile, which was comparable with that of the fenofibrate group, the AEs were significantly lower than in the fenofibrate group and an improvement in hepatobiliary enzyme activity was also recognized. However, we believe that actual clinical data as well as long-term efficacy and safety need to be investigated in the future.

Effects of lifestyle on hepatobiliary enzyme abnormalities following the Fukushima Daiichi nuclear power plant accident: The Fukushima health management survey.

Dramatic lifestyle changes due to the Fukushima Daiichi Nuclear Power Plant accident increased the prevalence of hepatobiliary enzyme abnormalities (HEA). We aimed to evaluate associations of HEA with specific lifestyle- and disaster-related factors in residents who lived near the Fukushima Daiichi Nuclear Power Plant.This cross-sectional study included 22,246 residents who underwent a Comprehensive Health Check and the Mental Health and Lifestyle Survey from June 2011 to March 2012. Residents were divided into 2 groups based on residential area and housing status after the accident. Associations between HEA and lifestyle- and disaster-related factors, including psychological distress, were estimated using logistic regression analysis adjusted for demographic and lifestyle factors.HEA was present in 27.3% of subjects. The prevalence of HEA was significantly higher in evacuees than controls (29.5% vs 25.7%, P < .001). There were significant differences in various lifestyle characteristics and the prevalence of post-traumatic stress disorder between evacuees and controls. Multivariable logistic regression analysis showed that age, sex, moderate to heavy drinking, and low/no physical activity were significantly associated with HEA regardless of evacuation status. Changes in jobs and unemployment were significantly associated with HEA in controls and evacuees, respectively.Lifestyle and disaster-related factors, but not psychological distress, were associated with HEA among subjects who lived near the Fukushima Daiichi Nuclear Power Plant accident.

Changes in Hepatobiliary Enzyme Abnormality After the Great East Japan Earthquake: The Fukushima Health Management Survey.

Although the incidence of hepatobiliary enzyme abnormality increased immediately after the Great East Japan Earthquake and subsequent Fukushima Daiichi Nuclear Power Plant accident, longer-term trends remain unclear. The aims of this study were to determine longer-term trends in hepatobiliary enzyme abnormality and to elucidate lifestyle factors associated with such changes among residents of a nuclear-disaster-affected area. This longitudinal survey enrolled 20,395 adults living in the vicinity of Fukushima Daiichi Nuclear Power Plant. Data were obtained from the records of annual health checkups of adults aged ≥40 years between 2011 and 2012. Follow-up examinations were conducted from June 2013 to March 2014. Associations were assessed between changes in hepatobiliary enzyme abnormality immediately and 3-4 years after the disaster and lifestyle factors. The overall prevalence of hepatobiliary enzyme abnormality significantly decreased over the study period, from 29.9% to 27.1%. Multivariate logistic regression analysis revealed significant associations between improved hepatobiliary enzyme abnormality and improvements in daily physical activity and frequency of breakfast consumption. The results suggest that improvements in daily physical activity and frequency of breakfast consumption significantly reduced the incidence of hepatobiliary enzyme abnormality 3-4 years after the Great East Japan Earthquake and Fukushima Daiichi Nuclear Power Plant accident.

Soluble Adenylyl Cyclase Regulates Bile Salt-Induced Apoptosis in Human Cholangiocytes.

UNLABELLED: Anion exchanger 2 (AE2), the principal bicarbonate secretor in the human biliary tree, is down-regulated in primary biliary cholangitis. AE2 creates a "bicarbonate umbrella" that protects cholangiocytes from the proapoptotic effects of bile salts by maintaining them deprotonated. We observed that knockdown of AE2 sensitized immortalized H69 human cholangiocytes to not only bile salt-induced apoptosis (BSIA) but also etoposide-induced apoptosis. Because the toxicity of etoposide is pH-independent, there could be a more general mechanism for sensitization of AE2-depleted cholangiocytes to apoptotic stimuli. We found that AE2 deficiency led to intracellular bicarbonate accumulation and increased expression and activity of soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. Thus, we hypothesized that sAC regulates BSIA. H69 cholangiocytes and primary mouse cholangiocytes were used as models. The sAC-specific inhibitor KH7 not only reversed sensitization to BSIA in AE2-depleted H69 cholangiocytes but even completely prevented BSIA. sAC knockdown by tetracycline-inducible short hairpin RNA also prevented BSIA. In addition, sAC inhibition reversed BSIA membrane blebbing, nuclear condensation, and DNA fragmentation. Furthermore, sAC inhibition also prevented BSIA in primary mouse cholangiocytes. Mechanistically, sAC inhibition prevented Bax phosphorylation at Thr167 and mitochondrial translocation of Bax and cytochrome c release but not c-Jun N-terminal kinase activation during BSIA. Finally, BSIA in H69 cholangiocytes was inhibited by intracellular Ca(2+) chelation, aggravated by thapsigargin, and unaffected by removal of extracellular calcium. CONCLUSIONS: BSIA is regulated by sAC, depends on intracellular Ca(2+) stores, and is mediated by the intrinsic apoptotic pathway; down-regulation of AE2 in primary biliary cholangitis sensitizes cholangiocytes to apoptotic insults by activating sAC, which may play a crucial role in disease pathogenesis. (Hepatology 2016;64:522-534).

Establishing benchmarks for the management of elevated liver enzymes and/or dilated biliary trees in an urban safety net hospital: analysis of 915 subjects.

BACKGROUND: The push for public reporting of outcomes necessitates relevant benchmarks for disease states across different settings. This study establishes benchmarks for choledocholithiasis management in a safety net hospital setting. METHODS: We reviewed all patients admitted to our acute care surgery service with biochemical evidence of choledocholithiasis who underwent same-admission cholecystectomy (CCY) between July 2012 and December 2013. RESULTS: During this 18-month period, 915 patients were admitted with biochemical evidence of choledocholithiasis. Descriptive statistics for the cohort are provided, which include a 51% rate of obesity and 95% rate of pathologic cholecystitis. Conversion rates of 4% and complication rates of 6% were found. The majority had a CCY without biliary imaging (n = 630, 68.9%). CONCLUSIONS: Relevant benchmarks are characterized, and results of a practice pattern of omitting pre- or intraoperative biliary tree imaging are described. These findings serve as a first benchmark of choledocholithiasis management for urban safety net hospitals.

Dipeptidylpeptidase-IV activity and expression reveal decreased damage to the intrahepatic biliary tree in fatty livers submitted to subnormothermic machine-perfusion respect to conventional cold storage.

Graft steatosis is a risk factor for poor initial function after liver transplantation. Biliary complications are frequent even after normal liver transplantation. A subnormothermic machine perfusion (MP20) preservation procedure was developed by our group with high potential for reducing injury to hepatocytes and sinusoidal cells of lean and fatty livers respect to conventional cold storage (CS). We report the response of the biliary tree to CS or MP20, in lean and obese Zucker rat liver. Dipeptidylpeptidase-IV (DPP-IV), crucial for the inactivation of incretins and neuropeptides, was used as a marker. Liver morphology and canalicular network of lean livers were similar after CS/reperfusion or MP20/reperfusion. CS preservation of fatty livers induced serious damage to the parenchyma and to the canalicular activity/expression of DPP-IV whereas with MP20 the morphology and canalicular network were similar to those of untreated lean liver. CS and MP20 had similar effects on DPP-IV activity and expression in the upper segments of the intrahepatic biliary tree of fatty livers. DPP-IV expression was significantly increased after MP20 respect to CS or to the controls, both for lean and obese animals. Our data support the superiority of MP20 over CS for preserving fatty livers. Dipeptidylpeptidase-IV activity and expression reveal decreased damage to the intrahepatic biliary tree in fatty livers submitted to subnormothermic machine-perfusion respect to conventional cold storage.

P450-mediated O-demethylated metabolite is responsible for rat hepatobiliary toxicity of pyridyltriazine-containing PI3K inhibitors.

The dysregulation of phosphatidylinositol 3-kinase (PI3K)-dependent pathways is implicated in several human cancers making it an attractive target for small molecule PI3K inhibitors. A series of potent pyridyltriazine-containing inhibitors of class Ia PI3Ks were synthesized and a subset of compounds was evaluated in exploratory repeat-dose rat toxicology studies. Daily oral dosing of compound 1: in Sprague Dawley rats for four consecutive days was associated with hepatobiliary toxicity that included biliary epithelial hyperplasia and hypertrophy, periductular edema, biliary stasis, and acute peribiliary inflammatory infiltrates. These histological changes were associated with clinical pathology changes that included increased serum liver enzymes, total bile acids, and bilirubin. The predominant clearance pathway of 1: was shown in vitro and in a bile-duct cannulated rat (14)C-ADME study to be P450-mediated oxidative metabolism. An O-demethylated pyridine metabolite, M3: , was identified as a candidate proximal metabolite that caused the hepatotoxicity. Co-administration of the pan-P450 inhibitor 1-aminobenzotriazole with 1: to rats significantly reduced the formation of M3: and prevented liver toxicity, whereas direct administration of M3: reproduced the toxicity. Structural changes were introduced to 1: to make the methoxypyridine ring less susceptible to P450 oxidation (compound 2: ), and addition of a methyl group to the benzylic carbon (compound 3: ) improved the pharmacokinetic profile. These changes culminated in the successful design of a clinical candidate 3: (AMG 511) that was devoid of liver toxicity in a 14-day rat toxicity study. Herein, we describe how a metabolism-based structure-activity relationship analysis allowed for the successful identification of a PI3K inhibitor devoid of off-target toxicity.

Prolonged, but transient, elevation of liver and biliary function tests in a healthy infant affected with breast milk jaundice.

Unconjugated hyperbilirubinaemia is a common finding in newborns. When it is exaggerated, it is usually investigated in order to exclude several diseases, such as newborn's haemolytic diseases, infections or hypothyroidism. Breast milk jaundice is a form of neonatal jaundice related to breast feeding and it is not usually associated with any clinical issue and/or other laboratory abnormalities. We describe a case of breast milk jaundice being associated, unexpectedly, to significant elevation of plasmatic liver and biliary enzymes. Despite the infant's good clinical condition and growth, several investigations were performed and these ruled out metabolic, infectious and autoimmune liver diseases. All liver function tests normalised by 6-7 months of life. We suggest that the finding of hypertransaminasaemia and hyper-γ-glutamyl transpeptidase in a benign clinical context (similar to what we described) should be followed for 6-7 months before performing sophisticated and expensive diagnostic investigations which aim at excluding some unlikely and severe diseases in a completely asymptomatic infant.

Metabolic enzyme activities and drug excretion in the small intestine and in the liver in the rat.

The aim of these experiments was the investigation of the correlation between the metabolic enzyme activities and the intestinal and hepatic excretion of p-nitrophenol (PNP) and its metabolites (PNP-glucuronide: PNP-G and PNP-sulfate: PNP-S) in the same group of rats (n = 10). A jejunal loop was perfused with isotonic medium containing PNP in a concentration of 500 µM. The samples were obtained from the luminal perfusion medium and from the bile. For enzyme assays tissue samples were obtained from the liver and jejunum at the end of experiments. Significant differences were calculated by the Student's t-test. The activity of UDP-glucuronyltransferase and sulfotransferase was about three times higher in the liver than in the small intestine. The activity of the ß-glucuronidase was about six times higher, the activity of the arylsulfatase was approximately seven times greater in the liver than in the jejunum. No significant difference was found between the luminal appearance and the biliary excretion of PNP-G. Contrary to these findings, the biliary excretion of PNP-S was significantly higher than the luminal appearance of PNP-sulfate. It can be concluded that no direct correlation exists between the activity of metabolic enzymes and the excretion rate of PNP-metabolites in the liver and in the jejunal segment of the small intestine.

Mutations in vacuolar H+ -ATPase subunits lead to biliary developmental defects in zebrafish.

We identified three zebrafish mutants with defects in biliary development. One of these mutants, pekin (pn), also demonstrated generalized hypopigmentation and other defects, including disruption of retinal cell layers, lack of zymogen granules in the pancreas, and dilated Golgi in intestinal epithelial cells. Bile duct cells in pn demonstrated an accumulation of electron dense bodies. We determined that the causative defect in pn was a splice site mutation in the atp6ap2 gene that leads to an inframe stop codon. atp6ap2 encodes a subunit of the vacuolar H(+)-ATPase (V-H(+)-ATPase), which modulates pH in intracellular compartments. The Atp6ap2 subunit has also been shown to function as an intracellular renin receptor that stimulates fibrogenesis. Here we show that mutants and morphants involving other V-H(+)-ATPase subunits also demonstrated developmental biliary defects, but did not demonstrate the inhibition of fibrogenic genes observed in pn. The defects in pn are reminiscent of those we and others have observed in class C VPS (vacuolar protein sorting) family mutants and morphants, and we report here that knockdown of atp6ap2 and vps33b had an additive negative effect on biliary development. Our findings suggest that pathways which are important in modulating intracompartmental pH lead to defects in digestive organ development, and support previous studies demonstrating the importance of intracellular sorting pathways in biliary development.

The evolution of the OATP hepatic uptake transport protein family in DMPK sciences: from obscure liver transporters to key determinants of hepatobiliary clearance.

Over the last two decades the impact on drug pharmacokinetics of the organic anion transporting polypeptides (OATPs: OATP-1B1, 1B3 and 2B1), expressed on the sinusoidal membrane of the hepatocyte, has been increasingly recognized. OATP-mediated uptake into the hepatocyte coupled with subsequent excretion into bile via efflux proteins, such as MRP2, is often referred to as hepatobiliary excretion. OATP transporter proteins can impact some drugs in several ways including pharmacokinetic variability, pharmacodynamic response and drug-drug interactions (DDIs). The impact of transporter mediated hepatic clearance is illustrated with case examples, from the literature and also from the Pfizer portfolio. The currently available in vitro techniques to study the hepatic transporter proteins involved in the hepatobiliary clearance of drugs are reviewed herein along with recent advances in using these in vitro data to predict the human clearance of compounds recognized by hepatic uptake transporters.

Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

Fascin is an actin-binding protein involved in the cell motility. Recently, aberrant expression of fascin in carcinoma cells was reported to participate in their invasive growth in cooperation with proteinases such as matrix metalloproteinases (MMPs). This study examined the participation of fascin in the progression of cholangiocarcinoma (CC) with reference to MMPs and tumor necrosis factor-alpha (TNF-alpha). Expression levels of fascin and MMP2 and 9 were examined immunohistochemically in human non-neoplastic biliary epithelium (13 cases) and CC (87 cases). The relationship between fascin and MMP9-expression levels was examined using two CC cell lines (CCKS-1 and HuCCT1). It was also examined whether or not fascin was involved in TNF-alpha-induced overproduction of MMP9 in CC. Fascin and MMP9 were expressed in 49 and 53% of CC samples, respectively, and the expression of these genes was frequent in intrahepatic CC. Fascin expression was correlated significantly with MMP9 expression. In particular, these two molecules were expressed more intensely at the invasive fronts of CC. Fascin expression was an unfavorable prognostic factor for patients with intrahepatic CC. In vitro studies showed that TNF-alpha could induce the overexpression of fascin and MMP9 in two CC cell lines. A knockdown study of fascin by siRNA showed that TNF-alpha induced the overproduction of fascin, which in turn upregulated MMP9 expression. Overexpression of fascin may have an important function in the progression of CC, and fascin expression might be involved in the signaling pathway in TNF-alpha-dependent production of MMP9 in CC.

Natural heme oxygenase-1 inducers in hepatobiliary function.

Many physiological effects of natural antioxidants, their extracts or their major active components, have been reported in recent decades. Most of these compounds are characterized by a phenolic structure, similar to that of alpha-tocopherol, and present antioxidant properties that have been demonstrated both in vitro and in vivo. Polyphenols may increase the capacity of endogenous antioxidant defences and modulate the cellular redox state. Changes in the cellular redox state may have wide-ranging consequences for cellular growth and differentiation. The majority of in vitro and in vivo studies conducted so far have attributed the protective effect of bioactive polyphenols to their chemical reactivity toward free radicals and their capacity to prevent the oxidation of important intracellular components. However, in recent years a possible novel aspect in the mode of action of these compounds has been suggested; that is, the ultimate stimulation of the heme oxygenase-1 (HO-1) pathway is likely to account for the established and powerful antioxidant/anti-inflammatory properties of these polyphenols. The products of the HO-catalyzed reaction, particularly carbon monoxide (CO) and biliverdin/bilirubin have been shown to exert protective effects in several organs against oxidative and other noxious stimuli. In this context, it is interesting to note that induction of HO-1 expression by means of natural compounds contributes to protection against liver damage in various experimental models. The focus of this review is on the significance of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against various stressors in several pathological conditions.

Diagnostic role of biliary pancreatic elastase for cholangiocarcinoma in patients with cholestasis.

BACKGROUND: A wide array of proteins is secreted into the bile and may be associated with biliary tract diseases. We attempted to discover novel biomarker in bile for cholangiocarcinoma. METHODS: Bile was collected from patients with bile duct obstruction. Proteins were separated by 2-dimensional electrophoresis and identified by mass spectrometry. Levels of mRNA and protein expression of the candidate biomarker were analyzed by real-time PCR and Western blotting, respectively, whereas enzyme activity was measured by a kinetic method. The diagnostic efficacy was assessed by receiver operating characteristic (ROC) curve analysis. RESULTS: Pancreatic elastase (PE) 3B was identified as a biomarker for cholangiocarcinoma. The mRNA of PE 3B was up-regulated in cancerous tissues, compared to non-cancerous tissues. The protein expression and enzyme activity of PE in bile were increased in patients with cholangiocarcinoma, compared to gallstone patients. Biliary amylase activity was used to correct the presence of pancreaticobiliary reflux. Significantly higher PE/amylase ratios in bile were found in patients with cholangiocarcinoma (0.214+/-0.045) than those with gallstone (0.023+/-0.005, p<0.001). The area under the ROC curve of the ratio was 0.877 (95% CI: 0.765 to 0.988). Using 0.065 as a cutoff value, the ratio distinguished malignant from benign causes of biliary obstruction with a sensitivity of 82% and a specificity of 89%. CONCLUSION: PE in bile is a biomarker for cholangiocarcinoma and the combination measurement of PE and amylase enhances diagnostic efficacy.

Bezafibrate may attenuate biliary damage associated with chronic liver diseases accompanied by high serum biliary enzyme levels.

BACKGROUND: Bezafibrate is a commonly used medicine for hyperlipidemia, and recently several reports have suggested the efficacy of bezafibrate for the treatment of primary biliary cirrhosis (PBC). To assess its efficacy for other liver diseases, we administered bezafibrate to patients with various categories of hepatobiliary impairment. METHODS: Bezafibrate (400 mg/day) was orally administered to 67 patients with chronic liver disease [22 with PBC, six with primary sclerosing cholangitis (PSC), 20 with chronic liver disease associated with hepatitis C virus (HCV) infection (CLD-C), seven with auto immune hepatitis (AIH), ten with alcoholic liver injury, and two with drug-induced liver injury]. RESULTS: The levels of biliary enzymes, such as alkaline phosphatase and gamma-glutamyltranspeptidase, decreased promptly and dramatically. The abnormally high level of alanine aminotransferase also showed a gradual decrease over 6 months in five of the eight PBC patients, all three PSC patients, eight of the 17 CLD-C patients, and all seven alcoholic liver injury patients. The level of immunoglobulin M showed a gradual decrease in 17 of the 22 PBC patients. CONCLUSIONS: Bezafibrate significantly reduced the level of biliary enzymes in various chronic liver diseases and may be useful for the treatment of certain liver disease subsets.

Transgenic mice aberrantly expressing pyruvate dehydrogenase complex E2 component on biliary epithelial cells do not show primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disorder that specifically destroys biliary epithelial cells (BECs). In patients with PBC, the immunodominant pyruvate dehydrogenase complex E2 component (PDC-E2), identified as an antigen for disease-specific anti-mitochondrial antibody, is expressed aberrantly in the BEC cytoplasm. The present study focused on the pathophysiological role of aberrant PDC-E2 in the development of PBC. The BEC-specific cytokeratin-19 promoter and PDC-E2 gene were cloned from a mouse cDNA library. The constructed transgene was microinjected into fertilized eggs of mice, and the offspring were identified by Southern blotting and reverse transcriptase-polymerase chain reaction. The protein expression was confirmed by immunoprecipitation, immunoblotting and immunohistochemical staining. Five founder lines were identified as carrying the PDC-E2 gene, and one of these lines expressed PDC-E2 mRNA. The protein expression of exogenous PDC-E2 was detected in the liver. The transgenic mouse line showed diffuse expression of PDC-E2 in the BEC cytoplasm. Biochemical, serological and histological features of PBC were not detected. We established transgenic mice that constitutively express PDC-E2. The results indicated that aberrant PDC-E2 expression in the cytoplasm of BECs is not sufficient for the initiation of autoimmunity. Additional factors may be required to establish a model of PBC.

In vitro detection of differential and cell-specific hepatobiliary toxicity induced by geldanamycin and 17-allylaminogeldanamycin using dog liver slices.

Experiments on rat liver slices demonstrated the differential hepatobiliary toxic potency of two anticancer agents, geldanamycin (GEL) and 17-allylaminogeldanamycin (17-AAG), over a 5-day period. This report describes the pattern of toxicity of these agents in dog liver tissue, using the in vitro liver slice culture model. Liver slices (200 microm thick) from male beagle dogs were cultured for 5 days in chemically defined culture medium containing a range of GEL and 17-AAG concentrations (0.1-5 microM). Tissues were evaluated using a panel of clinically relevant biomarkers and histological endpoints. GEL-induced reduction of alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) slice levels, indicators of biliary epithelial cell (BEC) viability, was supported by histological findings showing an increasing loss of BEC as higher concentrations were applied. At the highest concentrations studied, GEL caused both hepatocellular necrosis and BEC loss. Biomarker pattern results in the medium concurred with those from slice biochemistry measurements and histology. 17-AAG, a less potent compound in vivo, elicited more biomarker retention at higher concentrations than did GEL. Histological analysis revealed higher BEC viability and significant retention of BEC proliferation as compared with GEL. However, at the highest concentration, the toxic insult caused a marked decrease in BEC viability and proliferation. Comparison of responses with both compounds indicated that slices exposed to the same concentrations were more sensitive to GEL than to 17-AAG. Dog liver slices can thus be used to evaluate species-, compound-, and concentration-dependent differences in toxicity.

cAMP stimulates the secretory and proliferative capacity of the rat intrahepatic biliary epithelium through changes in the PKA/Src/MEK/ERK1/2 pathway.

BACKGROUND/AIMS: To evaluate if increased cholangiocyte cAMP levels alone are sufficient to enhance cholangiocyte proliferation and secretion. METHODS: Normal rats were treated in vivo with forskolin for two weeks. Cholangiocyte apoptosis, proliferation and secretion were evaluated. Purified cholangiocytes from normal rats were treated in vitro with forskolin in the absence or presence of Rp-cAMPs (a PKA inhibitor), PP2 (an Src inhibitor) or PD98059 (a MEK inhibitor). Subsequently, we evaluated cholangiocyte proliferation by determination of proliferating cellular nuclear antigen (PCNA) protein expression by immunoblots. We evaluated if the effects of forskolin on cholangiocyte functions are associated with changes in the cAMP/PKA/Src/MEK/ERK1/2 pathway. RESULTS: Chronic administration of forskolin to normal rats increased the number of ducts, cAMP levels, and secretin-induced choleresis compared to controls. Forskolin-induced increases in cholangiocyte proliferation and secretion were devoid of cholangiocyte necrosis, inflammation and apoptosis. In vitro, in pure isolated cholangiocytes, forskolin increased cholangiocyte proliferation, which was ablated by Rp-cAMPs, PP2 and PD98059. The effects of forskolin on cholangiocyte proliferation were associated with increased activity of PKA, Src Tyrosine 139 (Tyr 139) and ERK1/2. CONCLUSIONS: Modulation of the PKA/Src/MEK/ERK1/2 pathway may be important in the regulation of cholangiocyte growth and secretion observed in cholestatic liver diseases.