Toxic Bile Acids Research Articles

Primary biliary cirrhosis and bile acids

The dihydroxylated bile acid ursodeoxycholic acid (UDCA) has now been regarded for 20 years as the standard treatment for primary biliary cirrhosis (PBC), a chronic cholestatic immune-mediated condition marked by progressive destruction of small intrahepatic bile ducts, impaired biliary secretion, hepatocellular retention of toxic endogenous bile acids and, ultimately, the development of fibrosis leading to cirrhosis that commonly requires liver transplantation. At first sight, it seems intriguing that a bile acid could be considered for use as a therapeutic agent in a bile-acid secretion disorder. Yet, in addition to its inherently greater hydrophilic nature and competitive effect on endogenous bileacid recycling, UDCA has indeed been demonstrated to be a potent post-transcriptional secretagogue as well as a potential anti-inflammatory and anti-apoptotic agent. While the combined glucocorticoid receptor/pregnane X receptor (PXR) agonist budesonide, in combinaison with UDCA, has been shown to exert additional beneficial effects in PBC, significant progress in understanding the regulatory mechanisms involved in bile-acid homeostasis has led to the identification of nuclear [farnesoid X receptor (FXR), PXR, peroxisome proliferator-activated receptor alpha (PPARα)] and membrane (the membrane G protein-coupled bile acid receptor TGR5) receptors as critical pharmacological targets for future therapeutic approaches. Encouraging data from recent experimental and phase-II studies tend to confirm that the FXR agonist obeticholic acid and the PPARα agonists bezafibrate and fenofibrate may be used as add-on therapies in PBC patients with inadequate responses to UDCA or even as alternative first-line agents. These results could mark the beginning of a new therapeutic era for PBC.

Fibroblast growth factor 7 inhibits cholesterol 7α-hydroxylase gene expression in hepatocytes

Cholesterol 7α-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl4)-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

Albumin Regeneration for Extracorporeal Liver Support Using Prometheus: A Step in the Right Direction

Albumin Regeneration for Extracorporeal Liver Support Using Prometheus: A Step in the Right Direction

Consumption of Vitamin B6 Reduces Fecal Ratio of Lithocholic Acid to Deoxycholic Acid, a Risk Factor for Colon Cancer, in Rats Fed a High-Fat Diet

To examine the effect of supplemental dietary vitamin B(6) on the colonic luminal environment, growing male rats were fed a high-fat diet containing 1, 7, or 35 mg pyridoxine HCl/kg diet for 6 wk. Food intake and growth were unaffected by the dietary treatment. Supplemental dietary vitamin B(6) significantly reduced the production of a fecal secondary bile acid, lithocholic acid (the most toxic secondary bile acid and a risk factor for colon cancer), and markedly reduced the ratio of lithocholic acid to deoxycholic acid (a less toxic secondary bile acid) in feces (p<0.05). Increasing dietary vitamin B(6) increased fecal mucin levels (a marker of intestinal barrier function) in a dose-dependent manner (p<0.05) but did not affect fecal immunoglobulin A levels (an index of intestinal immune function). Cecal levels of organic acids were not significantly affected by supplemental dietary vitamin B(6). These results suggest the possibility that dietary vitamin B(6) affects the colonic luminal environment by altering the production of secondary bile acids and mucins.

Novel insight into mechanisms of cholestatic liver injury

Cholestasis results in a buildup of bile acids in serum and in hepatocytes. Early studies into the mechanisms of cholestatic liver injury strongly implicated bile acid-induced apoptosis as the major cause of hepatocellular injury. Recent work has focused both on the role of bile acids in cell signaling as well as the role of sterile inflammation in the pathophysiology. Advances in modern analytical methodology have allowed for more accurate measuring of bile acid concentrations in serum, liver, and bile to very low levels of detection. Interestingly, toxic bile acid levels are seemingly far lower than previously hypothesized. The initial hypothesis has been based largely upon the exposure of μmol/L concentrations of toxic bile acids and bile salts to primary hepatocytes in cell culture, the possibility that in vivo bile acid concentrations may be far lower than the observed in vitro toxicity has far reaching implications in the mechanism of injury. This review will focus on both how different bile acids and different bile acid concentrations can affect hepatocytes during cholestasis, and additionally provide insight into how these data support recent hypotheses that cholestatic liver injury may not occur through direct bile acid-induced apoptosis, but may involve largely inflammatory cell-mediated liver cell necrosis.

Protection against Cholestasis and Ischemia in vitro by Ursodeoxycholyl Lysophosphatidylethanolamide (UDCA-LPE) is Associated with Upregulation of Anti-apoptotic Proteins

Introduction: An increase of toxic bile acids such as glycochenodeoxycholic acid (GCDCA) frequently occurs in an early phase of liver transplantation. Ischemia during transplantation can cause hepatic cholestasis and damage of intrahepatic biliary epithelial cells. Both stresses contribute to the failure in liver transplantation. We aim to test the effectiveness of UDCA-LPE in inhibition of apoptosis induced by GCDCA and ischemia. Methods: Mouse hepatocytes were isolated and treated with 50µM GCDCA for 15h. As an experimental model of ischemia, CoCl2 at 500–750µM was used to treat the intrahepatic biliary epithelial cancer cell line MzChA1 for 24h. Protein expression of pro- and anti-apoptosis proteins and signalling molecules was measured by Western blot. The effect of UDCA-LPE on protein turnover was studied by immunoprecipitation and immunoblotting. Caspase 3 and 8 activities were measured by luminescence spectrometry. Results: In mouse hepatocytes and MzChA1, apoptosis was induced as measured by cleaved PARP–1, caspase 3 and 8 proteins and activities. Co-treatment with UDCA-LPE markedly inhibited GCDCA- and CoCl2-induced apoptosis. Expression of anti-apoptotic protein Mcl–1, cIAP2 and cFlip was decreased with CoCl2 treatment, and UDCA-LPE co-treatment prevented this decrease. By measuring ubiquitinated proteins, we found that UDCA-LPE inhibited the degradation of Mcl–1, but not of cIAP2 and cFlip proteins. This inhibition by UDCA-LPE was concomitant with phosphorylation of GSK–3α/β, which is a known mediator for Mcl–1 stabilization. Conclusion: The effect of UDCA-LPE on inhibition of Mcl–1 ubiquitination via phosphorylation of GSK–3α/β is one of the mechanisms for UDCA-LPE protection against apoptosis during bile acid and ischemic stresses. Further work is needed to investigate transcriptional control of cFlip and cIAP2 proteins by UDCA-LPE. UDCA-LPE may thus be used as an agent to prevent complications associated with liver transplantation.

A Biliary HCO3 − Umbrella Constitutes A Protective Mechanism Against Bile Acid–Induced Injury in Human Cholangiocytes

Human cholangiocytes are continuously exposed to millimolar levels of hydrophobic bile salt monomers. We recently hypothesized that an apical biliary HCO3- umbrella might prevent the protonation of biliary glycine-conjugated bile salts and uncontrolled cell entry of the corresponding bile acids, and that defects in this biliary HCO3- umbrella might predispose to chronic cholangiopathies. Here, we tested in vitro whether human cholangiocyte integrity in the presence of millimolar bile salt monomers is dependent on (1) pH, (2) adequate expression of the key HCO3- exporter, anion exchanger 2 (AE2), and (3) an intact cholangiocyte glycocalyx. To address these questions, human immortalized cholangiocytes and cholangiocarcinoma cells were exposed to chenodeoxycholate and its glycine/taurine conjugates at different pH levels. Bile acid uptake was determined radiochemically. Cell viability and apoptosis were measured enzymatically. AE2 was knocked down by lentiviral short hairpin RNA. A cholangiocyte glycocalyx was identified by electron microscopy, was enzymatically desialylated, and sialylation was quantified by flow cytometry. We found that bile acid uptake and toxicity in human immortalized cholangiocytes and cholangiocarcinoma cell lines in vitro were pH and AE2 dependent, with the highest rates at low pH and when AE2 expression was defective. An apical glycocalyx was identified on cholangiocytes in vitro by electron microscopic techniques. Desialylation of this protective layer increased cholangiocellular vulnerability in a pH-dependent manner. A biliary HCO3- umbrella protects human cholangiocytes against damage by bile acid monomers. An intact glycocalyx and adequate AE2 expression are crucial in this process. Defects of the biliary HCO3- umbrella may lead to the development of chronic cholangiopathies.

Effect of bile duct ligation on bile acid composition in mouse serum and liver

Cholestatic liver diseases can be caused by genetic defects, drug toxicities, hepatobiliary malignancies or obstruction of the biliary tract. Cholestasis leads to accumulation of bile acids (BAs) in hepatocytes. Direct toxicity of BAs is currently the most accepted hypothesis for cholestatic liver injury. However, information on which bile acids are actually accumulating during cholestasis is limited. To assess the BA composition in liver and serum after bile duct ligation (BDL) in male C57Bl/6 mice between 6h and 14days and evaluate toxicity of the most abundant BAs. Bile acid concentrations increased in liver (27-fold) and serum (1400-fold) within 6h after surgery and remained elevated up to 14days. BAs in livers of BDL mice became more hydrophilic than sham controls, mainly because of increased 6β-hydroxylation and taurine conjugation. Among the eight unconjugated and 16 conjugated BAs identified in serum and liver, only taurocholic acid (TCA), β-muricholic acid (βMCA) and TβMCA were substantially elevated representing >95% of these BAs over the entire time course. Although glycochenodeoxycholic acid and other conjugated BAs increased in BDL animals, the changes were several orders of magnitude lower compared with TCA, βMCA and TβMCA. A mixture of these BAs did not cause apoptosis or necrosis, but induced inflammatory gene expression in cultured murine hepatocytes. The concentrations of cytotoxic BAs are insufficient to cause hepatocellular injury. In contrast, TCA, βMCA and TβMCA are able to induce pro-inflammatory mediators in hepatocytes. Thus, BAs act as inflammagens and not as cytotoxic mediators after BDL in mice.

Hepatocyte IKK2 Protects Mdr2−/− Mice from Chronic Liver Failure

Mice lacking the Abc4 protein encoded by the multidrug resistance-2 gene (Mdr2 −/−) develop chronic periductular inflammation and cholestatic liver disease resulting in the development of hepatocellular carcinoma (HCC). Inhibition of NF-κB by expression of an IκBα super-repressor (IκBαSR) transgene in hepatocytes was shown to prevent HCC development in Mdr2 −/− mice, suggesting that NF-κB acts as a tumour promoter in this model of inflammation-associated carcinogenesis. On the other hand, inhibition of NF-κB by hepatocyte specific ablation of IKK2 resulted in increased liver tumour development induced by the chemical carcinogen DEN. To address the role of IKK2-mediated NF-κB activation in hepatocytes in the pathogenesis of liver disease and HCC in Mdr2 −/− mice, we generated Mdr2-deficient animals lacking IKK2 specifically in hepatocytes using the Cre-loxP system. Mdr2−/− mice lacking IKK2 in hepatocytes developed spontaneously a severe liver disease characterized by cholestasis, major hyperbilirubinemia and severe to end-stage fibrosis, which caused muscle wasting, loss of body weight, lethargy and early spontaneous death. Cell culture experiments showed that primary hepatocytes lacking IKK2 were more sensitive to bile acid induced death, suggesting that hepatocyte-specific IKK2 deficiency sensitized hepatocytes to the toxicity of bile acids under conditions of cholestasis resulting in greatly exacerbated liver damage. Mdr2−/−IKK2Hep-KO mice remarkably recapitulate chronic liver failure in humans and might be of special importance for the study of the mechanisms contributing to the pathogenesis of end-stage chronic liver disease or its implications on other organs. Conclusion: IKK2-mediated signaling in hepatocytes protects the liver from damage under conditions of chronic inflammatory cholestasis and prevents the development of severe fibrosis and liver failure.

Dose-Response of Five Bile Acids on Serum and Liver Bile Acid Concentrations and Hepatotoxicty in Mice

Feeding bile acids (BAs) to rodents has been used to study BA signaling and toxicity in vivo. However, little is known about the effect of feeding BAs on the concentrations of BAs in serum and liver as well as the dose of the fed BAs that causes liver toxicity. The present study was designed to investigate the relative hepatotoxicity of individual BAs by feeding mice cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), or ursodeoxycholic acid (UDCA) at concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, or 3% in their diet for 7 days. The data demonstrate that (1) the ability of the fed BAs to produce hepatotoxicity is UDCA<CA<CDCA<DCA<LCA; (2) the lowest concentration of each BA in the feed that causes hepatotoxicity in mice is CA and CDCA at 0.3%, DCA at 0.1%, and LCA at 0.03%; (3) BA feeding results in a dose-dependent increase in the total serum BA concentrations but had little effect on liver total BA concentrations; (4) hepatotoxicity of the fed BAs does not simply depend on the concentration or hydrophobicity of total BAs in the liver; and (5) liver BA-conjugation enzymes are saturated by feeding UDCA at concentrations higher than 0.3%. In conclusion, the findings of the present study provide guidance for choosing the feeding concentrations of BAs in mice and will aid in interpreting BA hepatotoxicity as well as BA-mediated gene regulation.

Profiling circulating and urinary bile acids in patients with biliary obstruction before and after biliary stenting.

Bile acids are considered as extremely toxic at the high concentrations reached during bile duct obstruction, but each acid displays variable cytotoxic properties. This study investigates how biliary obstruction and restoration of bile flow interferes with urinary and circulating levels of 17 common bile acids. Bile acids (conjugated and unconjugated) were quantified by liquid chromatography coupled with tandem mass spectrometry in serum and urine samples from 17 patients (8 men and 9 women) with biliary obstruction, before and after biliary stenting. Results were compared with serum concentrations measured in 40 age- and sex-paired control donors (20 men and 20 women). The total circulating bile acid concentration increases from 2.7 µM in control donors to 156.9 µM in untreated patients with biliary stenosis. Serum taurocholic and glycocholic acids exhibit 304- and 241-fold accumulations in patients with biliary obstruction compared to controls. The enrichment in chenodeoxycholic acid species reached a maximum of only 39-fold, while all secondary and 6α-hydroxylated species –except taurolithocholic acids – were either unchanged or significantly reduced. Stenting was efficient in restoring an almost normal circulating profile and in reducing urinary bile acids.ConclusionThese results demonstrate that biliary obstruction affects differentially the circulating and/or urinary levels of the various bile acids. The observation that the most drastically affected acids correspond to the less toxic species supports the activation of self-protecting mechanisms aimed at limiting the inherent toxicity of bile acids in face of biliary obstruction.

Profile of Serum Bile Acids in Noncholestatic Volunteers: Gender-Related Differences in Response to Fenofibrate

Fenofibrate belongs to hypolipidemic fibrates that act as activators of the peroxisome proliferator-activated receptor-α, a regulator of bile acid synthesis, metabolism and transport. The present study aimed at evaluating the effects of fenofibrate on the circulating bile acid profile in humans. Hundred healthy men and women completed a 3-week intervention with fenofibrate, and 17 bile acid species were measured in serum samples drawn before and after fenofibrate treatment. Fenofibrate caused significant reductions in levels of chenodeoxycholic (−26.4%), ursodeoxycholic (−30.5%), lithocholic (−18.4%), deoxycholic (−22.3%) and hyodeoxycholic (−19.2%) acids. A gender-related difference was observed in the response of various bile acids and the total bile acid concentration was significantly reduced only in men (−18.6%), while remaining almost unchanged in women (+0.36%). This difference detected suggests that fenofibrate should be more efficient at reducing bile acid toxicity in men than in women in cholestatic liver diseases.

ABC Transporters, Bile Acids, and Inflammatory Stress in Liver Cancer

The biliary secretion of bile acids is critical for multiple liver functions including digesting fatty nutrients and driving bile flow. When this process is impaired, the accumulating bile acids cause inflammatory liver injury. Multiple ABC transporters in the liver are key players to safeguard the hepatocyte and avoid toxicity due to bile acid over-accumulation. BSEP provides for efficient secretion of bile acids across the canalicular membrane against a steep concentration gradient. MDR3/Mdr2 and ABCG5/G8 secrete phosphatidylcholine and cholesterol, respectively, in coordination with BSEP-mediated bile acid secretion to mask the detergent/toxic effects of bile acids in the bile ductular space. Several lines of evidence indicate that when these critical steps are compromised, bile acid toxicity in vivo leads to inflammatory liver injury and liver cancer. In bsep-/- mice, liver cancer is rare. These mice display greatly increased expression of alternative bile acid transporters, such as Mdr1a/1b, Mrp3 and Mrp4. We believe these alternative transport systems provide an additional safeguard to avoid bile acid overload in liver. Such backup systems appear to be under-utilized in humans, as defects in BSEP and MDR3 lead to severe, often fatal childhood diseases. It is possible, therefore, that targeting ABC transporters and modulating the toxicity of the bile acid pool could be vital interventions to alleviate chronic inflammation and reduce the incidence of liver cancer in high-risk populations. The combination of an alternative ABC transporter with a novel substrate may prove an effective chemo-preventive or therapeutic strategy.

Changes of Organic Anion Transporter MRP4 and Related Nuclear Receptors in Human Obstructive Cholestasis

BackgroundHepatic multidrug resistance-associated protein 4 (Mrp4) levels are low, but increase markedly in rodent cholestatic liver. Nuclear receptors (NRs) are essential for regulating Mrp4 expression in cholestasis models. However, information about MRP4 and related NRs, including constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoic X receptor-α (RXRα), is relatively lacking in human obstructive cholestasis. We collected liver samples from patients with obstructive cholestasis or without liver disease and investigated the expression of MRP4 and NRs CAR, PXR, and RXRα by semi-quantitative RT-PCR, Western blot and immunostaining assays. ResultsMRP4 mRNA/protein levels were markedly increased in obstructive cholestasis. Concentration of serum total bile acids (TBA) was significantly correlated with MRP4 protein in cholestasis samples (P < 0.01). PXR and RXRα mRNA/protein levels were significantly increased in obstructive cholestasis. CAR mRNA levels were unchanged while protein levels were markedly induced in obstructive cholestasis. There was a statistically positive correlation between MRP4 mRNA and CAR protein (P < 0.05), suggesting that CAR may activate transcription of MRP4 genes by its nuclear translocation. ConclusionHepatic MRP4 levels were dramatically induced in human obstructive cholestasis, which may reduce liver injury by increasing efflux of toxic bile acids from hepatocytes into blood.

Nitric oxide mimics transcriptional and post-translational regulation during α-Tocopherol cytoprotection against glycochenodeoxycholate-induced cell death in hepatocytes

Reactive oxygen species (ROS) and nitric oxide (NO) exert a relevant role during bile acid-induced hepatotoxicity. Whether α-Tocopherol regulates oxidative and nitrosative stress, bile acid transporter expression and their NO-dependent post-translational modifications, and cell death were assessed in vitro and in vivo. α-Tocopherol and/or NO donors (DETA-NONOate or CSNO, and V-PYRRO/NO) were administered to glycochenodeoxycholic acid (GCDCA)-treated cultured human hepatocytes or to bile duct obstructed rats. Cell injury, superoxide anion (O⁻₂) production, as well as inducible nitric oxide synthase (NOS-2), cytochrome P4507A1 (CYP7A1), heme oxygenase-1, (HO-1) and bile acid transporter expression were determined. Cysteine S-nitrosylation and tyrosine nitration of Na(+)-taurocholate co-transporting polypeptide (NTCP), as well as taurocholic acid (TC) uptake were also evaluated. GCDCA-induced cell death was associated with increased (O⁻₂) production, NTCP and HO-1 expression, and with a reduction of CYP7A1 and NOS-2 expression. α-Tocopherol reduced cell death, (O⁻₂) production, CYP7A1, NTCP, and HO-1 expression, as well as increased NOS-2 expression and NO production in GCDCA-treated hepatocytes. α-Tocopherol and NO donors increased NTCP cysteine S-nitrosylation and tyrosine nitration, and reduced TC uptake in hepatocytes. α-Tocopherol and V-PYRRO/NO reduced liver injury and NTCP expression in obstructed rats. The regulation of CYP7A1, NTCP, and HO-1 expression may be relevant for the cytoprotective properties of α-Tocopherol and NO against mitochondrial dysfunction, oxidative stress and cell death in GCDCA-treated hepatocytes. The regulation of NO-dependent post-translational modifications of NTCP by α-Tocopherol and NO donors reduces the uptake of toxic bile acids by hepatocytes.

In vitro binding capacities of three dietary fibers and their mixture for four toxic elements, cholesterol, and bile acid

Water-soluble dietary fibers from apple peels and water-insoluble dietary fibers from wheat bran and soybean-seed hull were used to evaluate their binding capacities for four toxic elements (Pb, Hg, Cd, and As), lard, cholesterol, and bile acids. The water-soluble dietary fibers showed a higher binding capacity for three toxic cations, cholesterol, and sodium cholate; and a lower binding capacity for lard, compared to the water-insoluble ones. A mixture of the dietary fibers from all samples – apple peels, wheat bran, and soybean-seed hull – in the ratio 2:4:4 (w/w) significantly increased the binding capacity of water-insoluble dietary fibers for the three toxic cations, cholesterol, and sodium cholate; moreover, the mixture could lower the concentrations of Pb2+ and Cd+ in the tested solutions to levels lower than those occurring in rice and vegetables grown in polluted soils. However, all the tested fibers showed a low binding capacity for the toxic anion, AsO33−.

The Impact of Resuscitated Fecal Peritonitis on the Expression of the Hepatic Bile Salt Transporters in a Porcine Model

Sepsis is often associated with cholestatic liver dysfunction caused by changes in the expression profile of hepatic bile salt transporters. However, in rodent endotoxin models, the role of ischemic hepatitis caused by liver hypoperfusion cannot be delineated. We hypothesized that hepatocytes change their expression pattern of bile salt transporters during early severe sepsis despite adequate resuscitation. Fifteen anesthetized and instrumented pigs were randomized to either fecal peritonitis (n = 8) or control (n = 7). Resuscitation was performed by hydroxyethyl starch and norepinephrine infusion. Hemodynamic parameters and markers of cholestatic and ischemic hepatic dysfunction were recorded. At baseline and after 21 h, messenger RNA (mRNA) and protein expression of bile salt transporters was determined by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively, on in vivo liver biopsies. All resuscitated septic pigs developed a normotensive hyperdynamic circulation with increased portal flow. After 21 h of peritonitis, no signs of biochemical or histological cholestasis were present. Na-taurocholate cotransporting polypeptide and bile salt export pump mRNA were downregulated by 83% (P = 0.001) and 67% (P = 0.001), respectively, in comparison with controls, whereas multidrug resistance-associated protein 4 (MRP4) mRNA was upregulated by 85% (P = 0.02). Bile salt export pump and MRP2 staining were downregulated in septic pigs. During early porcine fluid-resuscitated sepsis, hepatic basolateral influx (Na-taurocholate cotransporting polypeptide) and canalicular efflux (bile salt export pump) of bile salts were downregulated without hemodynamic signs of hepatic hypoperfusion or biochemical signs of cholestasis. In parallel, the basolateral escape transport (MRP4) was markedly upregulated, possibly as an early adaptive response to counteract hepatocellular accumulation of toxic bile acids.

Partial bile duct ligation in mice: A novel model of acute cholestasis

The standard model for research in cholestasis is the total ligation of the bile duct (tBDL). Because this model causes severe hepatic injury in mice, we developed a novel model of cholestasis using a partial bile duct ligation (pBDL) and evaluate different mechanisms of injury. Male C57Bl/6 mice were subjected to sham operation, tBDL, or pBDL. Blood from tail veins was taken repeatedly until day 14 after surgery to assess markers of tissue injury (aspartate aminotransferase [AST]) and cholestasis (bilirubin, alkaline phosphatase [AP]). Also, liver samples were obtained at various time points to determine the histologic injury (hematoxylin and eosin) and tissue repair (Ki67). In addition, the biliary pressure and serum bile acids were evaluated as potential mechanisms of injury. Both models of cholestasis were equal in terms of bilirubin, AST, and AP serum levels during the first week of the experiment. Although these parameters remained constantly elevated thereafter in the tBDL model, all parameters normalized within the second week after pBDL. Moreover, pBDL resulted in significantly less necrosis formation (P = .001) and consequent hepatocyte proliferation (P= .01). Most important, serum bile acid levels (P = .04) and biliary pressures (P = .02) were significantly lower after pBDL than after tBDL and were the best predictors for hepatic necrosis formation. We established a model of acute cholestasis, which is ideal for research in resolved acute cholestasis (eg, surgery for Klatskin tumors). Moreover, biliary pressure and toxic bile acid serum levels may be better predictors of cholestatic liver injury than standard laboratory parameters.

Anti-oxidants do not prevent bile acid-induced cell death in rat hepatocytes

Bile acids, reactive oxygen species (ROS) and inflammatory cytokines are crucial regulators of cell death in acute and chronic liver diseases. The contribution of each factor to hepatocyte death, either apoptosis or necrosis, has not been clarified as yet. It has been suggested that the generation of oxidative stress by bile acids contributes to hepatocyte death during cholestasis and bile acid toxicity, although the beneficial role of ROS prevention in bile acid-mediated cell death is not fully understood. Study the effects of anti-oxidants in bile acid-induced cell death in vitro. Primary rat hepatocytes were exposed to the bile acids glycochenodeoxycholic acid (GCDCA) or taurolithocholic acid-3 sulphate in the absence or presence of ROS scavengers or anti-oxidants. Haeme oxygenase (HO)-1 mRNA levels were analysed by quantitative polymerase chain reaction. Apoptosis was quantified by acridine orange staining and caspase-3 activity assay. Necrosis was detected by Sytox green staining. Anti-oxidants do not attenuate bile acid-induced cell death. Furthermore, bile acid exposure does not enhance the mRNA expression of the oxidative stress-responsive gene HO-1. The Src-kinase inhibitor, SU6656, does reduce GCDCA-induced apoptosis and necrosis. In hepatocytes, bile acid-induced toxicity is not prevented by scavengers of oxidative stress. The beneficial effects observed in patients might be because of the contribution of ROS and cytokines rather than bile acid-mediated oxidative stress. However, the use of specific Src kinase inhibitors might be a useful tool to prevent bile acid-induced injury in liver diseases.

Sustained upregulation of sodium taurocholate cotransporting polypeptide and bile salt export pump and downregulation of cholesterol 7α-hydroxylase in the liver of patients with end-stage primary biliary cirrhosis

To examine the mRNA expression of hepatobiliary transporters in primary biliary cirrhosis (PBC) patients and to compare bile acid absorption, synthesis, and efflux in patients with non-end-stage and end-stage PBC, we obtained liver samples from PBC patients by percutaneous needle biopsy. End-stage PBC was defined as follows: histological stage IV; cirrhosis; serum total bilirubin, ≥4.0 mg/dl; and Child-Pugh Class C. The mRNA expression levels of sodium taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), and hepatic cholesterol 7α-hydroxylase (CYP7A1) were significantly higher in the PBC patients than in the controls (P < 0.01). The mRNA levels of NTCP and BSEP were significantly higher in the end-stage PBC patients than in the controls (P < 0.01). However, hepatic CYP7A1 mRNA expression decreased significantly (by 70%) in the patients with end-stage PBC as compared to the controls and the patients with non-end-stage PBC (P < 0.01). The hepatic expression of transporters mediating bile acid influx and efflux showed sustained elevation, whereas that of the rate-limiting enzyme for bile acid biosynthesis was attenuated in the end-stage PBC patients. Thus, mechanisms may be present preventing the accumulation of toxic bile acids in the hepatocytes of end-stage PBC patients.