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.

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.

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.

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.

Bile analysis as a tool for assessing integrity of biliary epithelial cells after cold ischemia--reperfusion of rat livers.

Previous morphological studies failed to show appreciable injury of biliary epithelial cells (BEC) after cold ischemia of rat liver, although recent evidence indicated that BEC integrity and function were impaired in this model. We tested the hypothesis that analysis of bile for enzymes, such as lactate dehydrogenase (LDH), alanine transaminase (ALT), and aspartate transaminase (AST), can be used for assessing cold ischemic injury of BEC. Furthermore, we examined whether biliary gamma-glutamyltransferase (GGT) reflects warm ischemic injury of BEC and whether normothermic reperfusion aggravates the negative effect of cold ischemia on BEC integrity and function. Rat livers were reperfused after different periods of cold or warm ischemia using a blood-free perfusion model. Compared with controls, perfusate LDH, ALT, and AST levels and parameters of hepatocyte function, including hepatocyte tight junction permeability, were not significantly altered by 18-h cold ischemia. On the other hand, 9-h cold ischemia markedly increased biliary LDH, ALT, and AST levels. However, only LDH release into the bile was strongly dependent on the time of cold storage. Biliary GGT, LDH, and glucose levels decreased during the reperfusion period following 18-h cold ischemia. The results suggest that biliary LDH can be used for assessing injury of BEC in cold-preserved livers and that normothermic reperfusion does not aggravate preservation-induced injury of BEC after cold ischemic storage.

Chemoprevention of biliary carcinogenesis in syrian hamsters by the novel carboxamide derivative IS-741 after initiation with N-nitrosobis(2-oxopropyl)amine (BOP).

To elucidate the possible inhibitory effect of a novel carboxamide derivative (IS-741) on biliary carcinogenesis, Syrian hamsters were subjected to cholecystoduodenostomy and ligation of the distal end of the common duct, and then given a regular diet (group I) or a diet containing 200 p.p.m. of IS-741 (group II). All hamsters were subcutaneously injected with N-nitrosobis(2-oxopropyl)amine until 10 weeks after surgery, and continued to feed on their respective dietary regimen until termination of the experiment at 16 weeks after surgery. Biliary adenocarcinomas were evaluated histologically. Non-cancerous and cancerous hepatobiliary tract tissues were analyzed for phospholipase A(2) (PLA(2)) activity, myeloperoxidase (MPO) activity, and the concentrations of prostaglandin (PG), i.e., prostaglandin E(2), 6-ketoprostaglandin F(1)alpha and thromboxane B(2). IS-741 significantly inhibited the development and multiplicity of hepatobiliary adenocarcinomas and reduced the proliferating cell nuclear antigen labeling indices in non-cancerous hepatobiliary tissues, compared with group I. The anti-cancerous effect of IS-741 was associated with a significant inhibition of PLA(2) and MPO levels in non-cancerous tissues of the extrahepatic biliary tract and the liver, and in cancerous tissue of the liver. Furthermore, IS-741 reduced the production of PGs in non-cancerous hepatobiliary tissues, compared with group I. Although the precise mechanism of action of IS-741 in preventing biliary tumorigenesis remains to be elucidated, it is likely to be related to modulation of arachidonic acid metabolism and/or suppression of neutrophil accumulation.

Phase I and phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium.

Tissue expression of drug-metabolizing enzymes influences susceptibility to drugs and carcinogens. Because the biliary epithelium, exposed to bile-borne chemicals, may give rise to drug-induced cholangiopathies and to cholangiocarcinomas, we determined the pattern of expression of drug-metabolizing enzymes in this epithelium. We first demonstrated by blot analyses that biliary epithelial cells (BEC) isolated from human gallbladders display cytochrome P450 (CYP) 1A, 2E1, and 3A, microsomal epoxide hydrolase (mEH), alpha, mu, and pi glutathione S-transferase (GST), transcripts and proteins. We also identified CYP-associated steroid 6beta-hydroxylase activity in BEC. CYP and mEH expression was 5- to 20-fold lower in BEC than in autologous hepatocytes, and further differed by a higher ratio of CYP3A5/CYP3A4, and by CYP1A1 predominance over CYP1A2. alphaGST was highly expressed in both hepatocytes and BEC, while piGST was restricted to BEC. In approximately 50% of individuals, muGST was expressed in hepatocytes and at lower levels in BEC. By using the same antibodies as those used in immunoblots, we could show by immunohistochemistry that CYP2E1, CYP3A, mEH, alpha, mu, and piGST immunoreactivities are expressed and display a heterogeneous distribution in the epithelium lining the entire biliary tract except for small intrahepatic bile ducts that were devoid of CYP3A and alphaGST immunoreactivities. In conclusion, BEC contribute to phase II, and although to a lesser extent than hepatocytes, to phase I biotransformation. The distribution of drug-metabolizing enzymes in BEC suggest that they are heterogeneous in their ability to generate and detoxicate reactive metabolites, which may contribute to specific distributions of cholangiopathies.

Immunohistochemical demonstration of beta-naphthoflavone-inducible cytochrome P450 1A1/1A2 in rat intrahepatic biliary epithelial cells.

Although intrahepatic biliary epithelial cells are targets for certain hepatotoxic chemicals, including some procarcinogens, their ability to monooxygenate, and thereby bioactivate and inactivate xenobiotics, remains to be established. Thus, the present study was undertaken to immunohistochemically determine if cytochrome P450 (CYP) 1A1/1A2 is present and can be induced within these nonparenchymal liver cells. Immunoperoxidase and immunofluorescent staining for CYP1A1/1A2 was detected within intrahepatic biliary epithelial cells as well as hepatocytes of control rats and was markedly enhanced in both cell types by beta-naphthoflavone (BNF). Color confocal laser microscopic analyses of dual immunofluorescent staining for CYP1A1/1A2 and cytokeratins 6 and 9 (56 and 64 kd, respectively) provided unequivocal evidence for the presence and induction of CYP1A1/1A2 within intrahepatic bile duct epithelia. Moreover, microdensitometric analyses of immunoperoxidase staining intensities for CYP1A1/1A2 revealed that intrahepatic biliary epithelial cells of control rats contain 44%, 56%, and 58% as much CYP1A1/1A2 as do centrilobular, midzonal, and periportal hepatocytes, respectively. These analyses further revealed that BNF increased the content of CYP1A1/1A2 in biliary epithelial cells by approximately 120%, while CYP1A1/1A2 levels in centrilobular, midzonal, and periportal hepatocytes were increased by 82%, 159%, and 160%, respectively. The results of this study represent the first in situ demonstration that mammalian intrahepatic biliary epithelial cells contain a CYP isoform, and further that CYP1A1/1A2 can be induced in these cells by BNF. These findings therefore indicate that intrahepatic biliary epithelial cells can oxidatively metabolize xenobiotics in situ and that their ability to bioactivate and inactivate xenobiotics can be significantly enhanced by CYP1A1/1A2 induction.

Inhibition of PDC-E2 human combinatorial autoantibodies by peptide mimotopes.

Immunohistochemical studies have shown that a unique immunoreactive molecule is present near the apical region of human biliary epithelial (BE) cells in patients with primary biliary cirrhosis (PBC). This can be visualized by confocal microscopy in PBC livers using a number of unique monoclonal antibodies to the E2 component of pyruvate dehydrogenase complex (PDC-E2), the autoantigen most commonly recognized by antimitochondrial antibodies (AMA). One such antibody, the murine mAb C355.1 was used to identify peptide mimotopes of PDC-E2 by screening a random dodecapeptide phage library ON 159.2 to identify the possible biochemical nature of this apical staining molecule. Out of 36 independent clones, 29 showed a common sequence and seven other sequences were singly represented. Three common amino acid motifs (SYP, TYVS and VRH) were found among these eight sequences. Similar to C355.1, the human combinatorial antibodies derived from a patient with PBC, SP1 and SP4, recognize the inner lipoyl domain of PDC-E2. However, when these antibodies are used to stain PBC BE cells, SP4 stains the apical region of PBC BE cells with high intensity whereas SP1 produces only cytoplasmic staining. Competitive inhibition of immunohistochemical staining using PDC-E2 specific human combinatorial antibodies SP1 and SP4 was performed using five of the above dodecapeptides. Interestingly, the peptides selected with C355.1 differentially inhibited the binding of SP1 and SP4 to PBC BE cells. Finally, rabbit sera raised against one such peptide (WMSYPDRTLRTS) stained BE cells from patients with PBC with a higher intensity than controls. Comparable data was obtained with immunoelectronmicroscopy. These data suggest that a molecular mimic of PDC-E2 is present at the external aspect of PBC BE cells.

A case of primary biliary cirrhosis associated with Hashimoto's thyroiditis, scleroderma and Sjögren's syndrome.

A 53-year-old woman was admitted because of Raynaud's phenomenon, polyarthralgia and polymyalgia. Biopsy specimens of the liver and thyroid gland revealed characteristic findings of primary biliary cirrhosis (PBC) (stage I by Scheuer's classification) and chronic thyroiditis. Her clinical features were also complicated by scleroderma (type I by Barnett's classification) and Sjögren's syndrome (Sjs) with keratoconjunctivitis sicca. Thyroid hormone replacement therapy led to improvement in thyroid function, normalization of the biliary tract enzymes and alleviation of subjective symptoms.

Effects of ATP depletion with DL-ethionine on biliary excretion of indocyanine green in the rat.

To clarify the energy-dependency of biliary excretion of a diagnostic dye, the effect of ATP depletion on biliary excretion was investigated. Biliary excretion of indocyanine green (ICG) in rat liver reached a maximum 20 min after intravenous application. The level of ATP in the liver was lowered to 35.4% of the control value about 5 h after administration of DL-ethionine (100 mg/100 g body weight). The rate of bile flow and the amount of ICG excreted in initial 10 min into the bile of ethionine-treated rats were lowered to 67.5% and 61.3% of the control values, respectively. The amount of ICG excreted for 10 min from 30 min to 40 min after application of ICG in ethionine-treated rats was comparable to that of the control. The level of ICG in blood 5 min after application of ICG was raised by 59.2% of the control value. These results suggest that ATP depletion in the liver caused by treatment with ethionine suppresses the initial rate of uptake and biliary excretion of ICG.

High-molecular-mass ("biliary") isoenzyme of alkaline phosphatase and the diagnosis of liver dysfunction in cystic fibrosis.

The high-Mr isoenzyme of alkaline phosphatase (AP, EC 3.1.3.1), a highly sensitive index to cholestasis, was measured by liquid chromatography in 45 patients with cystic fibrosis. Results of serum tests for liver dysfunction--including gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, total AP, bilirubin, and bile acids--were compared with those for high-Mr AP. Values for high-Mr AP were increased in 44.4% of our patient population, with activities ranging from 0.4 to 17.3 U/L. The upper limit in the control group was 2.5 U/L. We find increased high-Mr AP to be a more sensitive indicator of liver dysfunction in patients with cystic fibrosis than are other tests.