Epigenomic Evaluation of Cholangiocyte Transforming Growth Factor-ß Signaling Identifies a Selective Role for Histone 3 Lysine 9 Acetylation in Biliary Fibrosis.

BACKGROUND & AIMS: Transforming growth factor ß (TGFß) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFß that mediate transcription of fibrogenic signals. METHODS: Chromatin immunoprecipitation (ChIP)-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFß stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2-/- and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC)-fed mice were used as animal models. RESULTS: TGFß stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFß-mediated transcription. In contrast, H3K9ac was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFß. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFß-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFß-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis. CONCLUSIONS: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.

Protective role of cardiac-specific overexpression of caveolin-3 in cirrhotic cardiomyopathy.

Cirrhotic cardiomyopathy is a clinical syndrome in patients with liver cirrhosis characterized by blunted cardiac contractile responses to stress and/or heart rate-corrected QT (QTc) interval prolongation. Caveolin-3 (Cav-3) plays a critical role in cardiac protection and is an emerging therapeutic target for heart disease. We investigated the protective role of cardiac-specific overexpression (OE) of Cav-3 in cirrhotic cardiomyopathy. Biliary fibrosis was induced in male Cav-3 OE mice and transgene negative (TGneg) littermates by feeding a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; 0.1%) for 3 wk. Liver pathology and blood chemistries were assessed, and stress echocardiography, telemetry, and isolated heart perfusion studies to assess adrenergic responsiveness were performed. Cav-3 OE mice showed a similar degree of hyperdynamic contractility, pulmonary hypertension, and QTc interval prolongation as TGneg mice after 3 wk of DDC diet. Blunted systolic responses were shown in both DDC-fed Cav-3 OE and TGneg hearts after in vivo isoproterenol challenge. However, QTc interval prolongation after in vivo isoproterenol challenge was significantly less in DDC-fed Cav-3 OE hearts compared with DDC-fed TGneg hearts. In ex vivo perfused hearts, where circulatory factors are absent, isoproterenol challenge showed hearts from DDC-fed Cav-3 OE mice had better cardiac contractility and relaxation compared with DDC-fed TGneg hearts. Although Cav-3 OE in the heart did not prevent cardiac alterations in DDC-induced biliary fibrosis, cardiac expression of Cav-3 reduced QTc interval prolongation after adrenergic stimulation in cirrhosis.NEW & NOTEWORTHY Prevalence of cirrhotic cardiomyopathy is up to 50% in cirrhotic patients, and liver transplantation is the only treatment. However, cirrhotic cardiomyopathy is associated with perioperative morbidity and mortality after liver transplantation; therefore, management of cirrhotic cardiomyopathy is crucial for successful liver transplantation. This study shows cardiac myocyte specific overexpression of caveolin-3 (Cav-3) provides better cardiac contractile responses and less corrected QT prolongation during adrenergic stress in a cirrhotic cardiomyopathy model, suggesting beneficial effects of Cav-3 expression in cirrhotic cardiomyopathy.

A study of regulatory effects of TLR4 and NF-κB on primary biliary cholangitis.

OBJECTIVE: To investigate the regulatory effects of the Toll-like receptor 4 (TLR4) and the nuclear factor kappa-light-chain-enhancer of the activated B cells (NF-κB) on primary biliary cholangitis (PBC) and to analyze the possible mechanisms. MATERIALS AND METHODS: A total of 24 C57BL/6 mice were randomly divided into M group (n=12, intraperitoneally injected with polyinosinic acid-polycytidine acid (PolyI:C) for 12 consecutive weeks, 2 times/week) and C group (n=12, intraperitoneally injected with the same volume of normal saline). After 12 weeks, the mice were sacrificed to collect liver tissues. Then, an enzyme-linked immunosorbent assay (ELISA) kit was used to detect the content of interleukin-6 (IL-6), IL-10, and tumor necrosis factor-alpha (TNF-α) in liver tissues. Hematoxylin-eosin (HE) staining assay was performed to observe the pathological changes of liver tissues, and measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in peripheral blood of mice. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) staining was applied to determine cell apoptosis in liver tissues. The relative messenger ribonucleic acid (mRNA) expression levels of TLR4 and NF-κB in liver tissues were detected by quantitative Polymerase Chain Reaction (qPCR). Western blotting was adopted to measure the protein expressions of TLR4, NF-κB, myeloid differentiation factor 88 (MyD88), B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax), and Caspase-3. RESULTS: Compared with that in C group, the content of IL-6 and TNF-α in liver tissues in M group was significantly increased (p<0.01), but the level of IL-10 was statistically downregulated (p<0.01). According to HE staining, liver damage of mice in M group was evidently severer than that in C group, and the levels of ALT and AST in M group were significantly higher than those in C group (p<0.01). The amount of TUNEL-positive cells in liver tissues in M group was significantly greater than that in C group (p<0.01). The levels of TLR4 and NF-κB mRNA in liver tissues from M group were significantly elevated in comparison with the C group (p<0.01). Compared with those in C group, the expressions of TLR4, NF-κB, MyD88, and Caspase-3 proteins in M group showed statistical increases in liver tissues (p<0.01), whereas that of Bcl-2/Bax was significantly declined (p<0.01). CONCLUSIONS: PBC activates the TLR4/MyD88/NF-κB signaling pathway, induces the release of inflammatory factors and produces a large number of apoptotic proteins, which results in liver damage and cell apoptosis in mice.

3,5-Diethoxycarbonyl-1,4-Dihydrocollidine Diet: A Rodent Model in Cholestasis Research.

Liver cholestasis is characterized by impairment in bile flow. Among cholestatic diseases, primary biliary cholangitis and primary sclerosing cholangitis represent relevant causes of chronic liver disease, associated to significant morbidity and mortality. To better understand and to address therapeutic strategies to cholangiopathies is essential to develop an in vivo model which recapitulates the pathological features of the disease. Chronic feeding of 3,5-diethoxycarbonyl-1,4-dihydrocollidine, named DDC, has been proposed as an in vivo model for cholestatic disease due to the formation of intraductal porphyrin plugs. Chronic feeding of DDC in mice reproduces the main histopathological hallmarks of human cholestatic disease such as (1) remodeling of biliary compartments giving rise to ductular reaction, (2) periductular fibrosis, and (3) inflammatory infiltrate. This chapter describes the materials and methods necessary for the development and characterization of DDC diet-based mouse model.

Mesenchymal stem cells alleviate experimental autoimmune cholangitis through immunosuppression and cytoprotective function mediated by galectin-9.

BACKGROUND: Mesenchymal stem cells (MSCs) play an anti-inflammatory role by secreting certain bioactive molecules to exert their therapeutic effects for disease treatment. However, the underlying mechanism of MSCs in chronic autoimmune liver diseases-primary biliary cholangitis (PBC), for example-remains to be elucidated. METHODS: Human umbilical cord-derived MSCs (UC-MSCs) were injected intravenously into 2-octynoic acid coupled to bovine serum albumin (2OA-BSA)-induced autoimmune cholangitis mice. Serum levels of biomarkers and autoantibodies, histologic changes in the liver, diverse CD4+ T-cell subsets in different tissues, and chemokine activities were analyzed. Moreover, we investigated galectin-9 (Gal-9) expression and its function in UC-MSCs. RESULTS: In this study, UC-MSC transplantation (UC-MSCT) significantly ameliorated liver inflammation, primarily by diminishing T helper 1 (Th1) and Th17 responses as well as modifying liver chemokine activities in experimental autoimmune cholangitis mice. Mechanistically, UC-MSCs significantly repressed the proliferation of CD4+ T cells and suppressed the differentiation of Th1 and Th17 cells, which was likely dependent on Gal-9. Furthermore, the signal transducer and activator of transcription (STAT) and c-Jun N-terminal kinase (JNK) signaling pathways were involved in the production of Gal-9 in UC-MSCs. CONCLUSIONS: These results suggest that Gal-9 contributes significantly to UC-MSC-mediated therapeutic effects and improve our understanding of the immunomodulatory mechanisms of MSCs in the treatment of PBC.

Postprandial FGF19-induced phosphorylation by Src is critical for FXR function in bile acid homeostasis.

Farnesoid-X-Receptor (FXR) plays a central role in maintaining bile acid (BA) homeostasis by transcriptional control of numerous enterohepatic genes, including intestinal FGF19, a hormone that strongly represses hepatic BA synthesis. How activation of the FGF19 receptor at the membrane is transmitted to the nucleus for transcriptional regulation of BA levels and whether FGF19 signaling posttranslationally modulates FXR function remain largely unknown. Here we show that FXR is phosphorylated at Y67 by non-receptor tyrosine kinase, Src, in response to postprandial FGF19, which is critical for its nuclear localization and transcriptional regulation of BA levels. Liver-specific expression of phospho-defective Y67F-FXR or Src downregulation in mice results in impaired homeostatic responses to acute BA feeding, and exacerbates cholestatic pathologies upon drug-induced hepatobiliary insults. Also, the hepatic FGF19-Src-FXR pathway is defective in primary biliary cirrhosis (PBC) patients. This study identifies Src-mediated FXR phosphorylation as a potential therapeutic target and biomarker for BA-related enterohepatic diseases.

Dose-dependent effects of alpha-naphthylisothiocyanate disconnect biliary fibrosis from hepatocellular necrosis.

Exposure of rodents to the xenobiotic α-naphthylisothiocyanate (ANIT) is an established model of experimental intrahepatic bile duct injury. Administration of ANIT to mice causes neutrophil-mediated hepatocellular necrosis. Prolonged exposure of mice to ANIT also produces bile duct hyperplasia and liver fibrosis. However, the mechanistic connection between ANIT-induced hepatocellular necrosis and bile duct hyperplasia and fibrosis is not well characterized. We examined impact of two different doses of ANIT, by feeding chow containing ANIT (0.05%, 0.1%), on the severity of various liver pathologies in a model of chronic ANIT exposure. ANIT-elicited increases in liver inflammation and hepatocellular necrosis increased with dose. Remarkably, there was no connection between increased hepatocellular necrosis and bile duct hyperplasia and peribiliary fibrosis, as these pathologies increased similarly in mice exposed to either dose of ANIT. The results indicate that the severity of hepatocellular necrosis does not dictate the extent of bile duct hyperplasia/fibrosis in ANIT-exposed mice.

Fibrin deposition following bile duct injury limits fibrosis through an αMß2-dependent mechanism.

Coagulation cascade activation and fibrin deposits have been implicated or observed in diverse forms of liver damage. Given that fibrin amplifies pathological inflammation in several diseases through the integrin receptor αMß2, we tested the hypothesis that disruption of the fibrin(ogen)-αMß2 interaction in Fibγ(390-396A) mice would reduce hepatic inflammation and fibrosis in an experimental setting of chemical liver injury. Contrary to our hypothesis, α-naphthylisothiocyanate (ANIT)-induced liver fibrosis increased in Fibγ(390-396A) mice, whereas inflammatory cytokine expression and hepatic necrosis were similar to ANIT-challenged wild-type (WT) mice. Increased fibrosis in Fibγ(390-396A) mice appeared to be independent of coagulation factor 13 (FXIII) transglutaminase, as ANIT challenge in FXIII-deficient mice resulted in a distinct pathological phenotype characterized by increased hepatic necrosis. Rather, bile duct proliferation underpinned the increased fibrosis in ANIT-exposed Fibγ(390-396A) mice. The mechanism of fibrin-mediated fibrosis was linked to interferon (IFN)γ induction of inducible nitric oxide synthase (iNOS), a gene linked to bile duct hyperplasia and liver fibrosis. Expression of iNOS messenger RNA was significantly increased in livers of ANIT-exposed Fibγ(390-396A) mice. Fibrin(ogen)-αMß2 interaction inhibited iNOS induction in macrophages stimulated with IFNγ in vitro and ANIT-challenged IFNγ-deficient mice had reduced iNOS induction, bile duct hyperplasia, and liver fibrosis. Further, ANIT-induced iNOS expression, liver fibrosis, and bile duct hyperplasia were significantly reduced in WT mice administered leukadherin-1, a small molecule that allosterically enhances αMß2-dependent cell adhesion to fibrin. These studies characterize a novel mechanism whereby the fibrin(ogen)-integrin-αMß2 interaction reduces biliary fibrosis and suggests a novel putative therapeutic target for this difficult-to-treat fibrotic disease.

Statin pretreatment inhibits the lipopolysaccharide-induced epithelial-mesenchymal transition via the downregulation of toll-like receptor 4 and nuclear factor-κB in human biliary epithelial cells.

BACKGROUND AND AIM: Epithelial-mesenchymal transition (EMT) of biliary epithelial cells (BECs) plays an important role in biliary fibrosis. This study investigated the effects of simvastatin on the lipopolysaccharide (LPS)-induced EMT and related signal pathways in BECs. METHODS: Biliary epithelial cells were exposed to LPS (2 µg/mL) or transforming growth factor ß1 (TGF-ß1) (5 ng/mL) for 5 days. The EMT was assessed by a gain of mesenchymal cell markers (vimentin, N-cadherin, slug, and Twist-1) and a loss of epithelial cell markers (E-cadherin). The effects of simvastatin on the EMT induced by LPS or TGF-ß1 were determined by the changes in the levels of EMT markers and TLR4 and in the c-Jun N-terminal kinase (JNK), p38, and nuclear factor-κB (NF-κB) signaling pathways. RESULTS: Compared with the BECs treated with LPS alone, co-treatment with simvastatin and LPS induced an increase in the expression of E-cadherin and decreases in the expression levels of mesenchymal cell markers. The LPS-induced TLR4 expression level was slightly decreased by co-treatment with simvastatin. LPS-induced BEC growth was markedly inhibited by co-treatment with simvastatin. Furthermore, pretreatment with simvastatin inhibited the LPS-induced EMT in BECs by downregulating NF-κB and JNK phosphorylation. The suppressive effects of simvastatin pretreatment on the induction of the EMT by TGF-ß1 were also demonstrated in H69 cells. CONCLUSIONS: Our results demonstrate that LPS or TGF-ß1 promote the EMT in BECs that that pretreatment with simvastatin inhibited the induced EMT by downregulating toll-like receptor 4 and NF-κB phosphorylation. This finding suggests that simvastatin can be considered a new agent for preventing biliary fibrosis associated with the EMT of BECs.

Loss of keratin 19 favours the development of cholestatic liver disease through decreased ductular reaction.

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild-type (WT) and knockout (KO) mice were fed: (a) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline-deficient, ethionine-supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA- or CBDL-induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction.

Animal models of primary biliary cirrhosis.

Within the last decade, several mouse models that manifest characteristic features of primary biliary cirrhosis (PBC) with antimitochondrial antibodies (AMAs) and immune-mediated biliary duct pathology have been reported. Here, the authors discuss the current findings on two spontaneous (nonobese diabetic autoimmune biliary disease [NOD.ABD] and dominant negative transforming growth factor-ß receptor II [dnTGFßRII]) and two induced (chemical xenobiotics and microbial immunization) models of PBC. These models exhibit the serological, immunological, and histopathological features of human PBC. From these animal models, it is evident that the etiology of PBC is multifactorial and requires both specific genetic predispositions and environmental insults (either xenobiotic chemicals or microbial), which lead to the breaking of tolerance and eventually liver pathology. Human PBC is likely orchestrated by multiple factors and hence no single model can fully mimic the immunopathophysiology of human PBC. Nevertheless, knowledge gained from these models has greatly advanced our understanding of the major immunological pathways as well as the etiology of PBC.

Breach of tolerance: primary biliary cirrhosis.

In primary biliary cirrhosis (PBC), the breach of tolerance that leads to active disease involves a disruption in several layers of control, including central tolerance, peripheral anergy, a "liver tolerance effect," and the action of T regulatory cells and their related cytokines. Each of these control mechanisms plays a role in preventing an immune response against self, but all of them act in concert to generate effective protection against autoimmunity without compromising the ability of the host immune system to mount an effective response to pathogens. At the same time, genetic susceptibility, environmental factors, including infection agents and xenobiotics, play important roles in breach of tolerance in the development of PBC.

Aberrant TGF-ß1 signaling contributes to the development of primary biliary cirrhosis in murine model.

AIM: To investigate whether transforming growth factor-ß1 (TGF-ß1) signaling pathway is involved in the pathogenesis of primary biliary cirrhosis (PBC). METHODS: A murine model of PBC was developed by injection of polyinosinic polycytidylic acids (poly I: C) in C57BL/6 mice, and the liver expressions of TGF ß1, TGF-ß receptor I (TßRI), TGF-ß receptor II (TßRII), p-Smad2/3, monoclonal α-smooth muscle actin antibody (α-SMA) and α1 (I) collagen in the mouse model and control mice were evaluated by immunohistochemistry, immunoblotting and real-time polymerase chain reaction (RT-PCR). Lymphocyte subsets in liver were analyzed using flow cytometry. RESULTS: The mouse model had several key phenotypic features of human PBC, including elevated levels of alkaline phosphatase, antimitochondrial antibodies, portal bile ducts inflammation, and progressive collagen deposition. Compared with control mice, protein and mRNA levels of TGF ß1, TßRI, TßRII, p-Smad2/3, α-SMA and α1 (I) collagen in liver (1.7 ± 0.4 vs 8.9 ± 1.8, 0.8 ± 0.2 vs 5.1 ± 1.5, 0.6 ± 0.01 vs 5.1 ± 0.1, 0.6 ± 0.3 vs 2.0 ± 0.3, 0.9 ± 0.4 vs 3.4 ± 0.6, 0.8 ± 0.4 vs 1.7 ± 0.3, 1.1 ± 1.2 vs 11.8 ± 0.6, P < 0.05), and the total number and percentage of CD4⁺ CD25⁺ FOXP3⁺ and CD8⁺ lymphocytes (0.01 ± 0.001 vs 0.004 ± 0.00, 0.12 ± 0.04 vs 0.52 ± 0.23, P < 0.01) were higher in the mouse model. CONCLUSION: TGFß1 might play a dual role in the development of PBC: it suppresses inflammatory response but operates to enhance fibrogenesis. The aberrant activity of TGF-ß1 signaling contributes to the development of PBC.

Identification of potential cytokine pathways for therapeutic intervention in murine primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is considered a model autoimmune disease, with the most highly directed and specific autoantibody in both murine and human autoimmunity, the anti-mitochondrial autoantibody (AMA). However, therapeutic advances in this disease have lagged behind. Herein we have taken advantage of our unique model of murine PBC in which mice immunized with 2-octynoic acid coupled to BSA (2OA-BSA), a compound identified by quantitative structure activity relationships (QSAR) of human AMA binding, develop an intense inflammatory cholangitis with striking similarities to humans with PBC. In particular, we have constructed several unique gene-deleted mice, including mice deleted of IL-12p40, IL-12p35, IFN-γ, IL-23p19, IL-17A, IL-17F and IL-22, immunized these animals with 2OA-BSA and followed the natural history of immunopathology to identify key pathways that might provide clues for successful therapy. Our data indicate that whereas both IL-12/Th1 and IL-23/Th17 are involved in cholangitis, it is the IL-12/Th1 signaling pathway that elicits pathology. In fact, deletion of IFN-γ prevents disease and suppresses autoantibodies. Importantly, deletion of the Th17 cytokines IL-17A and IL-22, but not IL-17F, reduces biliary damage; IL-17A-knockout mice have reduced levels of anti-mitochondrial antibody. We further demonstrate that the production of IFN-γ is significantly decreased in the liver of IL-23p19(-/-), IL-17A(-/-) and IL-22(-/-) mice compared with controls. However, the ability of T cells to produce IFN-γ was not affected in Th17 cytokine-deficient mice. Our data indicate that a deficient Th17 pathway suppresses the accumulation of IFN-γ producing cells in liver during the early phase of cholangitis. In conclusion, whereas IFN-γ has a pivotal role in the early events involved in the pathogenesis of autoimmune cholangitis induced by 2OA-BSA, the IL-23/Th17 pathway potentiates the effects of IL-12/IFN-γ-mediated immunopathology.

Comparative proteomics study on liver mitochondria of primary biliary cirrhosis mouse model.

BACKGROUND: Primary biliary cirrhosis (PBC) is a liver specific chronic disease with unclear pathogenesis, especially for the early stage molecular events. The mitochondrion is a multi-functional organelle associated with various diseases including PBC. The purpose of this study was to discover the alterations in the mitochondria proteome using an early stage PBC mouse model for revealing the possible pathogenesis mechanisms in the early stages of PBC. METHODS: Mouse model of early stage of PBC was constructed by consecutive administration of poly I:C. Mitochondria of mouse models and controls were purified and comparative proteomics was performed by iTRAQ technology. Then, differentially expressed proteins were validated by western blotting. RESULTS: In total 354 proteins that satisfied the criteria for comparative proteomics study were identified. Of them, nine proteins were downregulated and 20 were up-regulated in liver mitochondria of PBC mouse model. Most differentially expressed proteins are associated with oxidation-reduction and lipid metabolism, and some are involved in the biosynthesis of steroid hormone and primary bile acid. Interestingly, four proteins (HCDH, CPT I, DECR, ECHDC2) involved in the fatty acid beta-oxidation were all upregulated. CONCLUSIONS: iTRAQ is a powerful tool for comparative proteomics study of PBC mouse model and differentially expressed proteins in mitochondria proteome of PBC mouse model provide insights for the pathogenesis mechanism at early stage of PBC.

Animal models of chronic liver diseases.

Chronic liver diseases are frequent and potentially life threatening for humans. The underlying etiologies are diverse, ranging from viral infections, autoimmune disorders, and intoxications (including alcohol abuse) to imbalanced diets. Although at early stages of disease the liver regenerates in the absence of the insult, advanced stages cannot be healed and may require organ transplantation. A better understanding of underlying mechanisms is mandatory for the design of new drugs to be used in clinic. Therefore, rodent models are being developed to mimic human liver disease. However, no model to date can completely recapitulate the "corresponding" human disorder. Limiting factors are the time frame required in humans to establish a certain liver disease and the fact that rodents possess a distinct immune system compared with humans and have different metabolic rates affecting liver homeostasis. These features account for the difficulties in developing adequate rodent models for studying disease progression and for testing new pharmaceuticals to be translated into the clinic. Nevertheless, traditional and new promising animal models that mimic certain attributes of chronic liver diseases are established and being used to deepen our understanding in the underlying mechanisms of distinct liver diseases. This review aims at providing a comprehensive overview of recent advances in animal models recapitulating different features and etiologies of human liver diseases.

Xenobiotics and autoimmunity: does acetaminophen cause primary biliary cirrhosis?

The serologic hallmark of primary biliary cirrhosis (PBC) is the presence of antimitochondrial autoantibodies (AMAs) directed against the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). The PBC-related autoepitope of PDC-E2 contains lipoic acid, and previous work has demonstrated that mimics of lipoic acid following immunization of mice lead to a PBC-like disease. Furthermore, approximately one-third of patients who have ingested excessive amounts of acetaminophen (paracetamol) develop AMA of the same specificity as patients with PBC. Quantitative structure-activity relationship (QSAR) data indicates that acetaminophen metabolites are particularly immunoreactive with AMA, and we submit that in genetically susceptible hosts, electrophilic modification of lipoic acid in PDC-E2 by acetaminophen or similar drugs can facilitate a loss of tolerance and lead to the development of PBC.

Tartrazine and sunset yellow are xenoestrogens in a new screening assay to identify modulators of human oestrogen receptor transcriptional activity.

Primary biliary cirrhosis (PBC) is a cholestatic liver disease of unknown cause that occurs most frequently in post-menopausal women. Since the female sex hormone oestrogen can be cholestatic, we hypothesised that PBC may be triggered in part by chronic exposure to xenoestrogens (which may be more active on a background of low endogenous oestrogen levels seen in post-menopausal women). A reporter gene construct employing a synthetic oestrogen response element predicted to specifically interact with oestrogen receptors (ER) was constructed. Co-transfection of this reporter into an ER null cell line with a variety of nuclear receptor expression constructs indicated that the reporter gene was trans-activated by ERα and ERß, but not by the androgen, thyroid, progesterone, glucocorticoid or vitamin D receptors. Chemicals linked to PBC were then screened for xenoestrogen activity in the human ERα-positive MCF-7 breast cancer cell line. Using this assay, the coal-derived food and cosmetic colourings--sunset yellow and tartrazine--were identified as novel human ERα activators, activating the human ER with an EC(50%) concentration of 220 and 160 nM, respectively.

Xenobiotic-induced liver injury and fibrosis.

INTRODUCTION: Many different drugs and xenobiotics (chemical compounds foreign to an organism) can injure the bile duct epithelium and cause inflammatory bile duct diseases (cholangiopathies) ranging from transient cholestasis to vanishing bile duct syndrome, sclerosing cholangitis with development of biliary fibrosis and cirrhosis. Animal models of xenobiotic-induced liver injury have provided major mechanistic insights into the molecular mechanisms of xenobiotic-induced cholangiopathies and biliary fibrosis including primary biliary cirrhosis and primary sclerosing cholangitis. AREAS COVERED: In this review, the authors discuss the basic principles of xenobiotic-induced liver and bile duct injury and biliary fibrosis with emphasis on animal models. A PubMed search was performed using the search terms "xenobiotic," "liver injury," "cholestasis," and "biliary fibrosis." Reference lists of retrieved articles were also searched for relevant literature. EXPERT OPINION: Xenobiotic-induced cholangiopathies are underestimated and frequently overlooked medical conditions due to their often transient nature. However, biliary disease may progress to vanishing bile duct syndrome, biliary fibrosis, and cirrhosis. Moreover, xenobiotics may prime the liver for subsequent liver disease by other agents and may also contribute to the development of hepatobiliary cancer though interaction with resident stem cells.