A systemic mechanism of increased transendothelial migration of leukocytes through the blood-brain barrier in hepatic encephalopathy.

BACKGROUND: Hepatic encephalopathy (HE) is a frequent neurological complication of cirrhosis. Evidence suggests a synergic pathophysiological implication of hyperammonemia and systemic inflammation. In addition, the blood-brain barrier (BBB) permeability can be impaired in cirrhotic patients, notably in those displaying HE. We hypothesized that systemic inflammation could trigger leukocytes transendothelial migration (TEM) through the BBB in cirrhotic patients and especially those with HE. METHODS: We studied the effects of patients' plasma on the TEM of the leukocyte U937 cell line in vitro, using a validated BBB model (hCMEC/D3 cell line). We compared TEM of U937 leukocytes across hCMEC/D3 monolayer incubated with the plasma of i) patients with cirrhosis without HE, ii) patients with cirrhosis and HE, iii) healthy controls. RESULTS: We show that the plasma of cirrhotic patients with HE enhances TEM of U937 leukocytes across hCMEC/D3 BBB model. We found a correlation between U937 TEM on the one hand, the West-Haven score and ammonemia on the other one. A trend towards a correlation between U937 TEM and PS-100Beta in plasma, a marker of BBB solute's permeability increase, was also found. CONCLUSION: These findings suggest that circulating factors could increase leukocytes TEM in cirrhotic patients and contribute to the increased BBB permeability that has been described in cirrhotic patients with HE.

ATP-binding cassette transporters expression in rats with cirrhosis and hepatic encephalopathy.

BACKGROUND: Pathophysiology of acute encephalopathy in cirrhotic patients is not completely understood. Factors implicated include ammonia, inflammation, various metabolic disorders and drug toxicity. Recent studies have evidenced an increased permeability of the blood-brain barrier (BBB) in models of chronic liver disease and encephalopathy, either to solutes, or to leukocytes. A modification of the expression of BBB ATP-Binding Cassette (ABC) transporters, actively transporting endogenous and exogenous components through the BBB, has been described in models of acute liver failure. We hypothesized that a modification of ABC transporters expression may contribute to drug-induced acute encephalopathy in cirrhosis. MATERIEL AND METHODS: A rat model of cirrhosis induced by Bile Duct Ligation (BDL) was studied, and compared to a SHAM rat model. Rats were sacrificed and brains studied after decapitation. Genic expression of ABC transporters, including P-gp, BCRP, MRP1, MRP2, MRP4 and MRP5 was evaluated by RT-qPCR on isolated brain microvessels. Encephalopathy was assessed 6 weeks after surgery by a trail suspension test and an Open Field Test. RESULTS: BDL rats developed a histologically proven cirrhosis and displayed a higher ammonemia than SHAM rats (183 µmol/L vs 53 µmol/L, p = 0.0003). BDL rats presented with encephalopathy shown by neurobehavioral tests. MRP2 was not detected neither in BDL nor in SHAM rats. There was a decrease in the genic expression of MRP5 6 weeks after surgery. Expressions of P-gp, BCRP, MRP1 and MRP4 were not different between the 2 groups. CONCLUSION: We suggest that acute encephalopathy in cirrhotic BDL rats may be associated to a modification of ABC transporter MRP5 on the BBB, that could be responsible for a decrease clearance of neurotoxic agents.

Diagnostic accuracy of the Coopscore© to predict liver fibrosis in human immunodeficiency virus/hepatitis B virus co-infection.

Background Non-invasive methods for assessing liver fibrosis are increasingly used as an alternative to liver biopsy. Recently, a score-based biochemical blood test (Coopscore©) was developed in a cohort of patients chronically infected with hepatitis C virus, showing higher diagnostic performances than Fibrometer®, Fibrotest®, Hepascore® and Fibroscan™. Here, we assess its performance in patients co-infected with the human immunodeficiency virus and hepatitis B virus. Methods Ninety-seven human immunodeficiency virus/hepatitis B virus co-infected patients with liver biopsies were included from a previously described cohort. Histological fibrosis staging using METAVIR criteria was used as the reference. Coopscore©, Fibrotest®, Fibrometer®, Hepascore® and Zeng score were computed and compared with the Coopscore© using the Obuchowski index and area under the receiving operator characteristic curves. Results The distribution of liver fibrosis levels was as follows: F0-F1 ( n = 42), F2 ( n = 25), F3 ( n = 15) and F4 ( n = 15). The Obuchowski index was higher for Coopscore© (0.774) than Fibrometer® (0.668), Hepascore® (0.690) and Zeng scores (0.704) ( P < 0.05), reflecting a better ability to discriminate between fibrosis stages. Similarly, when predicting significant fibrosis (≥F2), the AUROC was significantly greater for the Coopscore© (0.836) than the Hepascore® (0.727) and Zeng scores (0.746), but not for the Fibrotest® (0.778, P = 0.14) or Fibrometer® (0.790, P = 0.19). The Coopscore© did not show a higher capacity than other scores to predict advanced fibrosis (≥F3) or cirrhosis (F4). Conclusions This study supports the diagnostic value of the Coospcore© in fibrosis staging among human immunodeficiency virus/hepatitis B virus co-infected patients, especially to predict significant fibrosis.

AST/ALT ratio is not an index of liver fibrosis in chronic hepatitis C when aminotransferase activities are determinate according to the international recommendations.

OBJECTIVE: The aspartate aminotransferase activity (AST)/alanine aminotransferase activity (ALT) ratio is used as liver fibrosis index whereas the reported data are conflicting. In chronic hepatitis C (CHC), reported diagnostic accuracies range from none to good for significant fibrosis and to excellent for cirrhosis. Assuming that AST/ALT increases are mainly due to vitamin B6 defects since pyridoxal phosphate (PLP), active form of B6, acts as coenzyme in transamination reactions, we evaluated the diagnostic accuracy of the AST/ALT ratio using standardized methods for AST and ALT activities, with PLP addition as recommended, in a prospective multicenter cohort of CHC patients. METHODS: ALT and AST activities were measured using the recommended IFCC methods with addition of pyridoxal 5'-phosphate. We evaluated the AST/ALT ratio for the diagnosis of liver fibrosis or cirrhosis in a cohort of CHC patients included in a multicenter prospective study. A liver biopsy was performed in each patient and reviewed by two independent pathologists in order to determine the fibrosis stage according to Metavir classification which was the reference standard. RESULTS: AST/ALT ratio significantly increased with histological stage of liver fibrosis and there was a significant correlation between Metavir fibrosis stage and AST/ALT ratio (r=0.129, P<0.0035). The ROC curve analyses showed that the AST/ALT ratio does not discriminate significant fibrosis (F≥2) (AUROC=0.531) and had only very poor diagnostic accuracies for severe fibrosis (F≥3) (AUROC=0.584) or cirrhosis (F4) (AUROC=0.626). CONCLUSION: AST/ALT ratio is not a good and discriminative index of liver fibrosis in CHC when aminotransferase activities are determinate according to the international recommendations.

Vitamin D nuclear receptor deficiency promotes cholestatic liver injury by disruption of biliary epithelial cell junctions in mice.

UNLABELLED: Alterations in apical junctional complexes (AJCs) have been reported in genetic or acquired biliary diseases. The vitamin D nuclear receptor (VDR), predominantly expressed in biliary epithelial cells in the liver, has been shown to regulate AJCs. The aim of our study was thus to investigate the role of VDR in the maintenance of bile duct integrity in mice challenged with biliary-type liver injury. Vdr(-/-) mice subjected to bile duct ligation (BDL) displayed increased liver damage compared to wildtype BDL mice. Adaptation to cholestasis, ascertained by expression of genes involved in bile acid metabolism and tissue repair, was limited in Vdr(-/-) BDL mice. Furthermore, evaluation of Vdr(-/-) BDL mouse liver tissue sections indicated altered E-cadherin staining associated with increased bile duct rupture. Total liver protein analysis revealed that a truncated form of E-cadherin was present in higher amounts in Vdr(-/-) mice subjected to BDL compared to wildtype BDL mice. Truncated E-cadherin was also associated with loss of cell adhesion in biliary epithelial cells silenced for VDR. In these cells, E-cadherin cleavage occurred together with calpain 1 activation and was prevented by the silencing of calpain 1. Furthermore, VDR deficiency led to the activation of the epidermal growth factor receptor (EGFR) pathway, while EGFR activation by EGF induced both calpain 1 activation and E-cadherin cleavage in these cells. Finally, truncation of E-cadherin was blunted when EGFR signaling was inhibited in VDR-silenced cells. CONCLUSION: Biliary-type liver injury is exacerbated in Vdr(-/-) mice by limited adaptive response and increased bile duct rupture. These results indicate that loss of VDR restricts the adaptation to cholestasis and diminishes bile duct integrity in the setting of biliary-type liver injury.

Including osteoprotegerin and collagen IV in a score-based blood test for liver fibrosis increases diagnostic accuracy.

BACKGROUND: Noninvasive methods for liver fibrosis evaluation in chronic liver diseases have been recently developed, i.e. transient elastography (Fibroscan™) and blood tests (Fibrometer®, Fibrotest®, and Hepascore®). In this study, we aimed to design a new score in chronic hepatitis C (CHC) by selecting blood markers in a large panel and we compared its diagnostic performance with those of other noninvasive methods. METHODS: Sixteen blood tests were performed in 306 untreated CHC patients included in a multicenter prospective study (ANRS HC EP 23 Fibrostar) using METAVIR histological fibrosis stage as reference. The new score was constructed by non linear regression using the most accurate biomarkers. RESULTS: Five markers (alpha-2-macroglobulin, apolipoprotein-A1, AST, collagen IV and osteoprotegerin) were included in the new function called Coopscore©. Using the Obuchowski Index, Coopscore© shows higher diagnostic performances than for Fibrometer®, Fibrotest®, Hepascore® and Fibroscan™ in CHC. Association between Fibroscan™ and Coopscore© might avoid 68% of liver biopsies for the diagnosis of significant fibrosis. CONCLUSION: Coopscore© provides higher accuracy than other noninvasive methods for the diagnosis of liver fibrosis in CHC. The association of Coopscore© with Fibroscan™ increases its predictive value.

Comparison of nine blood tests and transient elastography for liver fibrosis in chronic hepatitis C: the ANRS HCEP-23 study.

BACKGROUND & AIMS: Blood tests and transient elastography (Fibroscan™) have been developed as alternatives to liver biopsy. This ANRS HCEP-23 study compared the diagnostic accuracy of nine blood tests and transient elastography (Fibroscan™) to assess liver fibrosis, vs. liver biopsy, in untreated patients with chronic hepatitis C (CHC). METHODS: This was a multicentre prospective independent study in 19 French University hospitals of consecutive adult patients having simultaneous liver biopsy, biochemical blood tests (performed in a centralized laboratory) and Fibroscan™. Two experienced pathologists independently reviewed the liver biopsies (mean length=25±8.4 mm). Performance was assessed using ROC curves corrected by Obuchowski's method. RESULTS: Fibroscan™ was not interpretable in 113 (22%) patients. In the 382 patients having both blood tests and interpretable Fibroscan™, Fibroscan™ performed similarly to the best blood tests for the diagnosis of significant fibrosis and cirrhosis. Obuchowski's measure showed Fibrometer® (0.86), Fibrotest® (0.84), Hepascore® (0.84), and interpretable Fibroscan™ (0.84) to be the most accurate tests. The combination of Fibrotest®, Fibrometer®, or Hepascore® with Fibroscan™ or Apri increases the percentage of well classified patients from 70-73% to 80-83% for significant fibrosis, but for cirrhosis a combination offers no improvement. For the 436 patients having all the blood tests, AUROC's ranged from 0.82 (Fibrometer®) to 0.75 (Hyaluronate) for significant fibrosis, and from 0.89 (Fibrometer® and Hepascore®) to 0.83 (FIB-4) for cirrhosis. CONCLUSIONS: Contrarily to blood tests, performance of Fibroscan™ was reduced due to uninterpretable results. Fibrotest®, interpretable Fibroscan™, Fibrometer®, and Hepascore® perform best and similarly for diagnosis of significant fibrosis and cirrhosis.

Liver fibrosis changes in HIV-HBV-coinfected patients: clinical, biochemical and histological effect of long-term tenofovir disoproxil fumarate use.

BACKGROUND: Data on liver fibrosis evolution in HIV-HBV-coinfected patients treated with tenofovir disoproxil fumarate (TDF) are scarce. The effect of TDF on liver fibrosis in 148 HIV-HBV-coinfected patients was prospectively evaluated using Fibrometer∆ scores and liver biopsies in a subset of patients. METHODS: The mean change from baseline (Δ) in Fibrometer score was modelled using a generalized estimating equation. Homogeneous continuous-time Markov models were used to study risk factors for regression or progression of liver fibrosis. RESULTS: Median follow-up of patients treated with TDF was 29.5 months (25th-75th percentile 20.9-38.1). The distribution of scored fibrosis at TDF initiation was F0-F1 n=65, F2 n=37 and F3-F4 n=46. In patients with a baseline fibrosis score of F3-F4, Fibrometer score decreased with a triphasic shape (Fibrometer Δ at 12, 24 and 36 months after TDF initiation was -0.079, -0.069 and -0.102, respectively). Despite duration on TDF, higher fibrosis scores were noted in F3-F4 patients with high HBV viral load and HDV coinfection, and in F0-F2 patients who had high HBV viral load and low CD4(+) T-cell count. Progression in fibrosis score over time was influenced by age, alcohol consumption, low CD4(+) T-cell count and HCV coinfection, whereas HDV coinfection and longer duration of HBV infection prevented fibrosis regression. No influence of antiretrovirals other than TDF was found. CONCLUSIONS: The use of TDF in HIV-HBV-coinfected patients led to a decrease in liver fibrosis score in patients with advanced fibrosis or cirrhosis. Sustainability of its direct antiviral and indirect antifibrotic effects on the liver need to be studied further.

Automation of the Hepascore and validation as a biochemical index of liver fibrosis in patients with chronic hepatitis C from the ANRS HC EP 23 Fibrostar cohort.

BACKGROUND: Hepascore combining serum bilirubin, gamma glutamyl transpeptidase, hyaluronic acid (HA) and alpha2-macroglobulin with age and sex, was reported as relevant in predicting liver fibrosis in patients with chronic HCV infection and was proposed as an alternative to liver biopsy. METHODS: Since an automated HA assay (Latex method, Wako, Japan) became available, we investigated to automate Hepascore by simultaneous measurements of components using an OLYMPUS AU640 analyzer (Tokyo, Japan). For its clinical evaluation, we considered a cohort of chronic HCV patients included in a multicenter prospective study (ANRS HC EP 23 Fibrostar). RESULTS: Automated Hepascore was not significantly different than assayed as previously described. An improvement in HA variability was evidenced. In 512 chronic HCV patients, automated Hepascore, using ROC curves analysis, showed good predictive performances for significant fibrosis (AUROC=0.81), severe fibrosis (AUROC=0.82), and cirrhosis (AUROC=0.88). For significant fibrosis, Hepascore (cut-off=0.5) had a sensitivity of 0.77, a specificity of 0.70, a positive predictive value of 0.71 and a negative predictive value (NPV) of 0.77. Hepascore <0.25 could exclude significant fibrosis with a sensitivity of 0.95 and a NPV of 0.90 and Hepascore <0.75 could exclude cirrhosis with a sensitivity of 0.86 and a NPV of 0.97. CONCLUSIONS: This study shows that Hepascore, a non-invasive index of liver fibrosis, necessitating only one serum sample, can be totally automated using a single analyzer and confirms that Hepascore accurately predicts liver fibrosis in chronic HCV. Hepascore might be largely used in assessing liver fibrosis as surrogate to the liver biopsy.

Impact of nutrition on phenotype in CFTR-deficient mice.

To elucidate the impact of nutrition in cystic fibrosis (CF), we compared the phenotypic traits of Cftr -/- mice fed either a lipid-enriched liquid diet (Peptamen) or a standard chow combined with polyethylenglycol osmotic laxative (PEG), two strategies commonly used to prevent intestinal obstruction in CF mice. Survival, growth, liver, and ventilatory status were determined in Cftr -/- and Cftr +/+ mice, followed-up until 120 d. Ventilation was recorded in conscious animals using whole-body plethysmography. We found that the survival rate was similar in Peptamen and PEG Cftr -/- mice. Cftr -/- mice had lower minute ventilation than Cftr +/+ mice, whatever the diet. Both Cftr -/- and Cftr +/+ mice fed Peptamen displayed preadult growth delay compared with PEG-treated animals. Despite subsequent growth catch-up, Cftr -/- mice remained smaller than Cftr +/+ mice, whatever the diet. All Peptamen fed Cftr -/- mice showed hepatomegaly and liver steatosis, which also occurred but to a lesser extent in Peptamen fed Cftr +/+ animals. Therefore, while both treatment strategies are similarly efficient to avoid high mortality at weaning, Peptamen induces preadult growth delay and liver steatosis. These effects of diet are important to consider in future animal studies and also prompt to evaluate high-energy diets in CF patients.

Diffusion-weighted magnetic resonance imaging for the assessment of fibrosis in chronic hepatitis C.

UNLABELLED: Liver biopsy is the gold standard for assessing fibrosis but has several limitations. We evaluated a noninvasive method, so-called diffusion-weighted magnetic resonance imaging (DWMRI), which measures the apparent diffusion coefficient (ADC) of water, for the diagnosis of liver fibrosis in patients with chronic hepatitis C virus (HCV). We analyzed 20 healthy volunteers and 54 patients with chronic HCV (METAVIR: F0, n = 1; F1, n = 30; F2, n = 8; F3, n = 5; and F4, n = 10) prospectively included. Patients with moderate-to-severe fibrosis (F2-F3-F4) had hepatic ADC values lower than those without or with mild fibrosis (F0-F1; mean: 1.10 +/- 0.11 versus 1.30 +/- 0.12 x 10(-3) mm2/s) and healthy volunteers (mean: 1.44 +/- 0.02 x 10(-3) mm2/s). In discriminating patients staged F3-F4, the areas under the receiving operating characteristic curves (AUCs) were 0.92 (+/-0.04) for magnetic resonance imaging (MRI), 0.92 (+/-0.05) for elastography, 0.79 (+/-0.08) for FibroTest, 0.87 (+/-0.06) for the aspartate aminotransferase to platelets ratio index (APRI), 0.86 (+/-0.06) for the Forns index, and 0.87 (+/-0.06) for hyaluronate. In these patients, the sensitivity, specificity, positive predictive value, and negative predictive value were 87%, 87%, 72%, and 94%, respectively, with an ADC cutoff level of 1.21 x 10(-3) mm2/s. In discriminating patients staged F2-F3-F4, the AUC values were 0.79 (+/-0.07) for MRI, 0.87 (+/-0.05) for elastography, 0.68 (+/-0.09) for FibroTest, 0.81 (+/-0.06) for APRI, 0.72 (+/-0.08) for the Forns index, and 0.77 (+/-0.06) for hyaluronate. CONCLUSION: This preliminary study suggests that DWMRI compares favorably with other noninvasive tests for the presence of significant liver fibrosis.

Hypoxia-induced changes in the expression of rat hepatobiliary transporter genes.

Cholestatic disorders may arise from liver ischemia (e.g., in liver transplantation) through various mechanisms. We have examined the potential of hypoxia to induce changes in the expression of hepatobiliary transporter genes. In a model of arterial liver ischemia subsequent to complete arterial deprivation of the rat liver, the mRNA levels of VEGF, a hypoxia-inducible gene, were increased fivefold after 24 h. The pattern of VEGF-induced expression and ultrastructural changes, including swelling of the endoplasmic reticulum, indicated that hypoxia affected primarily cholangiocytes, but also hepatocytes, predominantly in the periportal area. Serum and bile analyses demonstrated liver dysfunction of cholestatic type with reduced bile acid biliary excretion. Fluorescence-labeled ursodeoxycholic acid used as a tracer displayed no regurgitation, eliminating bile leakage as a significant mechanism of cholestasis in this model. In liver tissue, a marked reduction in the mRNA levels of Na(+)-taurocholate-cotransporting polypeptide (Ntcp), bile salt export protein (Bsep), and multidrug resistance-associated protein 2 (Mrp2) and an increase in those of Cftr were detected before bile duct proliferation occurred. In cultured hepatocytes, a nontoxic hypoxic treatment caused a decrease in the mRNA and protein expression of Ntcp, Bsep, and Mrp2 and in the mRNA levels of nuclear factors involved in the transactivation of these genes, i.e., HNF4alpha, RXRalpha, and FXR. In bile duct preparations, hypoxic treatment elicited an increase in Cftr transcripts, along with a rise in cAMP, a major regulator of Cftr expression and function. In conclusion, hypoxia triggers a downregulation of hepatocellular transporters, which may contribute to cholestasis, whereas Cftr, which drives secretion in cholangiocytes, is upregulated.

Chronic liver injury during obstructive sleep apnea.

Patients with obstructive sleep apnea (OSA) are at risk for the development of fatty liver as a result of being overweight. Several data suggest that OSA per se could be a risk factor of liver injury; ischemic hepatitis during OSA has been reported, and OSA is an independent risk factor for insulin resistance. Therefore, we investigated liver damage and potential mechanisms in 163 consecutive nondrinking patients with nocturnal polysomnographic recording for clinical suspicion of OSA. Serum levels of liver enzymes were measured in all patients. Liver biopsy was offered to patients with elevated liver enzymes. Intrahepatic hypoxia was assessed by the expression of vascular endothelial growth factor (VEGF) on liver biopsy specimens. Severe OSA (apnea-hypopnea index [AHI] > 50/hr) was seen in 27% of patients; 52% had moderate OSA (AHI 10-50/hr), and 21% had no OSA. Overall, 20% had elevated liver enzymes. Independent parameters associated with elevated liver enzymes were body mass index (BMI) (OR: 1.13; CI: 1.03-1.2) and severe OSA (OR: 5.9; CI: 1.2-29). Liver biopsy was performed in 18 of 32 patients with elevated liver enzymes and showed steatohepatitis in 12 cases, associated with fibrosis in 7 cases. Patients with severe OSA were more insulin-resistant according to homeostasis model assessment, had higher percentage of steatosis as well as scores of necrosis and fibrosis, despite similar BMI. Hepatic immunostaining used as an indirect marker of hypoxia was not different between patients with or without severe OSA. In conclusion, severe OSA is a risk factor for elevated liver enzymes and steatohepatitis independent of body weight. Promotion of insulin resistance is probably involved. Further studies are needed to determine whether hypoxia contributes directly to liver injury.

Adaptative bile duct proliferative response in experimental bile duct ischemia.

BACKGROUND/AIMS: A rat model of bile duct ischemia was established and used to examine the potential of bile duct proliferation to provide an adaptative response in cholestatic disorders. METHODS: Rats underwent partial or complete arterial deprivation of the liver. Serum biochemical tests, histological analyses and bile secretion measurements were performed at different time points up to 6 weeks after surgery. RESULTS: Rats developed biochemical signs of cholestasis exclusively after complete arterial deprivation. Within 4h, cholangiocytes in these rats showed morphological signs of cell damage. After 48h, they displayed VEGF expression and became proliferative. The proportion of Ki67-labeled cholangiocytes ( approximately 30%) was similar in interlobular bile ducts and periportal ductules. A ductular reaction made of well-formed bile ducts confined to portal tracts developed within 1 week. Bile flow which was initially decreased, was restored at 3 weeks, while the biochemical signs of cholestasis completely resolved at 6 weeks. At this time, the number of bile duct sections was maximal. Fibrosis intensity was also maximal, although moderate (

Contribution of mrp2 in alterations of canalicular bile formation by the endothelin antagonist bosentan.

BACKGROUND/AIMS: Bosentan, a dual endothelin ET(A/B) receptor antagonist, may cause dose-dependent reversible cholestatic liver injury. We herein tested whether bosentan or metabolites, both eliminated in bile, induce alterations in bile secretion. METHODS: Bile flow and output of bile constituents were monitored in pentobarbital-anesthetized rats with biliary fistulas. Normal and TR(-) rats with a genetic defect in mrp2, received bosentan intravenous injections. RESULTS: Bosentan bolus intravenous injections of 0.1-10mg/kg triggered a dose-dependent increase in biliary bilirubin excretion. In addition, doses (> or =10mg/kg) caused a sustained increase in canalicular bile salt-independent bile flow, combined with significant increases in the concentration and output of glutathione and of bicarbonate in bile. In rats receiving bosentan (> or =10mg/kg), both under basal conditions and under intravenous taurocholate perfusion (2micromol/min/kg), phospholipid and cholesterol secretions were profoundly inhibited and uncoupled from bile salt secretion. In TR(-) rats, the choleretic effect of bosentan was reduced to non-significant levels. The stimulation of bilirubin secretion and the uncoupling of phospholipid from bile salt secretion were absent, whereas that of cholesterol was maintained. CONCLUSIONS: Bosentan alters canalicular bile formation in major part via mrp2-mediated mechanisms. Intermittent uncoupling of lipid from bile salt secretion may contribute to bosentan hepatic adverse reaction.