Bile Salt Export Pump Research Articles

Measures of BSEP Inhibition In Vitro Are Not Useful Predictors of DILI.

Inhibition of the bile salt export pump (BSEP) by a drug has been implicated as a risk factor for a drug's potential to cause drug-induced liver injury (DILI) and is thought to be an important mechanism leading to DILI. For a wide variety of drugs a correlation has been observed between the potency of in vitro BSEP inhibition and its propensity to cause DILI in humans. These findings were interpreted to suggest that BSEP inhibition could be an important mechanism to help explain how some drugs initiate DILI. Because the Biopharmaceutics Drug Disposition Classification System (BDDCS) can be useful in characterizing and predicting some important transporter effects in terms of drug-drug interactions, we evaluated the information provided by BDDCS in order to understand the inhibition propensity of BSEP. Here we analyze the relationship between a compound's ability to inhibit BSEP function and cause liver injury in humans using a compilation of published DILI datasets that have screened for BSEP inhibitors, other hepatic transporters and other mechanism-based toxicity key events. Our results demonstrate that there is little support for in vitro BSEP inhibition being universally DILI predictive. Rather we show that most potent BSEP inhibitors are BDDCS class 2 drugs, which we have demonstrated previously is the BDDCS class most likely to be DILI related. Since BDDCS class is not related to any proposed DILI mechanistic hypotheses, we maintain that if measures of BSEP inhibition alone or together with inhibition of other transporters cannot be differentiated from class 2 assignment, there is no support for in vitro BSEP inhibition being DILI predictive.

Phytosterols Synergize With Endotoxin to Augment Inflammation in Kupffer Cells but Alone Have Limited Direct Effect on Hepatocytes.

Phytosterols are implicated in the development of parenteral nutrition-associated liver disease. A newly proposed mechanism for phytosterol-mediated parenteral nutrition-associated liver disease is through phytosterol-facilitated hepatic proinflammatory cytokine release following exposure to intestinally derived bacteria. Whether the proinflammatory effects are liver cell specific is not known. To determine if phytosterols cause inflammation in hepatocytes or Kupffer cells independently or require costimulation by lipopolysaccharide (LPS). In an in vivo study, neonatal piglets on parenteral nutrition for 11 days received an 8-hour infusion of LPS. In the in vitro studies, neonatal piglet Kupffer cells and hepatocytes were treated with media, media + 1% soy oil, or media + 1% soy oil + 100µM phytosterols. After 24-hour incubation, cells were treated with farnesoid X receptor (FXR) agonist obeticholic acid or liver X receptor (LXR) agonist GW3965 and challenged with LPS or interleukin 1β. LPS administration in piglets led to transient increases in proinflammatory cytokines and suppression of the transporters bile salt export pump and ATP-binding cassette transporter G5. In hepatocytes, phytosterols did not activate inflammation. Phytosterol treatment alone did not activate inflammation in Kupffer cells but, combined with LPS, synergistically increased interleukin 1β production. FXR and LXR agonists increased transporter expression in hepatocytes. GW3965 suppressed proinflammatory cytokine production in Kupffer cells, but obeticholic acid did not. LPS suppresses transporters that control bile acid and phytosterol clearance. Phytosterols alone do not cause inflammatory response. However, with costimulation by LPS, phytosterols synergistically maximize the inflammatory response in Kupffer cells.

Galactosylated Pro-Drug of Ursodeoxycholic Acid: Design, Synthesis, Characterization, and Pharmacological Effects in a Rat Model of Estrogen-Induced Cholestasis.

Ursodeoxycholic acid (UDCA) is considered the first-choice therapy for cholestatic disorders. To enhance solubility and exploit specific transporters in liver, we synthesized a new galactosyl pro-drug of UDCA (UDCAgal). Ethinylestradiol (EE)-induced cholestasis was used to study and compare the effects of UDCAgal with UDCA on bile flow, hepatic canalicular efflux transporter expression, and inflammation. UDCAgal resulted quite stable both at pH 7.4 and 1.2 and regenerated the parent drug after incubation in human plasma. Its solubility, higher than UDCA, was pH- and temperature-independent. UDCAgal displayed a higher cell permeation compared to UDCA in liver HepG2 cells. Moreover, in cholestatic rats, UDCAgal showed a higher potency compared to UDCA in reducing serum biomarkers (AST, ALT, and ALP) and cytokines (TNF-α and IL-1β). The higher effect of UDCAgal on the increase in bile salt export pump and multidrug resistance-associated protein 2 transcription indicated an improved spillover of bile acids from the liver. UDCAgal showed a reduction in CCL2, as well as TNF-α, IL-1β, and cyclooxygeanse-2 mRNAs, indicating a reduction in hepatic neutrophil accumulation and inflammation. Moreover, UDCAgal, similarly to UDCA, heightens bile flow and modulates biliary acids secretion. These results indicate that UDCAgal has a potential in the treatment of cholestatic disease.

Individual serum bile acid profiling in rats aids in human risk assessment of drug-induced liver injury due to BSEP inhibition

Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2hours (h) after dosing and returned to baseline values before 24h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.

Alteration of Bile Acid and Cholesterol Biosynthesis and Transport by Perfluorononanoic Acid (PFNA) in Mice.

Perfluorochemicals produce hepatotoxic effects via activation of peroxisome proliferator-activated receptor alpha (PPARα) and constitutive androstane receptor (CAR) nuclear receptors in animals. Bile formation is one major liver function. But it remains unknown whether perfluorochemicals alter metabolism of bile acids (BAs) in liver. The present study was designed to determine the impact of perfluorononanoic acid (PFNA) on BA and cholesterol homeostasis in mice. A single dose of PFNA (0.1 mmol/kg) was intraperitoneally administered to adult male wild-type (WT), PPARα-null, and CAR-null mice. PFNA caused cholestasis in the WT mice, indicated by increased serum alanine aminotransferase, hyperbilirubinemia, elevated BA concentrations in mouse serum, and appearance of bile plugs in mouse liver. In addition, PFNA decreased total and some individual BAs in mouse liver. PFNA increased the concentrations of total and taurine-conjugated, as well as some individual BAs in the serum of WT and CAR-null mice but not in PPARα-null mice, indicating a PPARα-dependent mechanism. PFNA decreased mRNA expression of most BA-related transporters (sodium-taurocholate cotransporting polypeptide, organic anion transporting polypeptide [Oatp]1a1, Oatp1b2, and bile salt export pump) and BA biosynthetic enzymes (Cyp7a1, 7b1, 8b1, and 27a1) in mouse liver, but increased mRNA expression of some efflux transporters (breast cancer resistance protein, multidrug resistance transporter 2, multidrug resistance-associated protein [Mrp] 2, Mrp3, and Mrp4), primarily via a PPARα-dependent mechanism. Moreover, PFNA increased free and total cholesterol in mouse liver but not in mouse serum. Furthermore, PFNA increased mRNA expression of sterol transporters, namely Abca1, g1, g5/g8, and steroidogenic acute regulatory protein via PPARα. In conclusion, PFNA produced cholestasis in mouse liver, and the activation of PPARα plays a central role in regulating BA and cholesterol metabolism and transport in mouse serum and liver.

Inhibition of Bile Salt Export Pump (BSEP) in Relation to Systemic Exposure: A Risk Factor for Drug Induced Liver Injury (DILI)

Inhibition of Bile Salt Export Pump (BSEP) in Relation to Systemic Exposure: A Risk Factor for Drug Induced Liver Injury (DILI)

Activation of insulin-like growth factor 1 receptor participates downstream of GPR30 in estradiol-17β-D-glucuronide-induced cholestasis in rats.

Estradiol-17β-D-glucuronide (E17G), through the activation of different signaling proteins, induces acute endocytic internalization of canalicular transporters in rat, including multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11), generating cholestasis. Insulin-like growth factor 1 receptor (IGF-1R) is a membrane-bound tyrosine kinase receptor that can potentially interact with proteins activated by E17G. The aim of this study was to analyze the potential role of IGF-1R in the effects of E17G in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets. In vitro, IGF-1R inhibition by tyrphostin AG1024 (TYR, 100nM), or its knock-down with siRNA, strongly prevented E17G-induced impairment of Abcc2 and Abcb11 function and localization. The protection by TYR was not additive to that produced by wortmannin (PI3K inhibitor, 100nM), and both protections share the same dependency on microtubule integrity, suggesting that IGF-1R shared the signaling pathway of PI3K/Akt. Further analysis of the activation of Akt and IGF-1R induced by E17G indicated a sequence of activation GPR30-IGF-1R-PI3K/Akt. In IPRL, an intraportal injection of E17G triggered endocytosis of Abcc2 and Abcb11, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of Abcc2 and Abcb11 substrates. TYR did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of transporters into the canalicular membrane. In conclusion, the activation of IGF-1R is a key factor in the alteration of canalicular transporter function and localization induced by E17G, and its activation follows that of GPR30 and precedes that of PI3K/Akt.

Adenovirus-mediated human aquaporin-1 expression in hepatocytes improves lipopolysaccharide-induced cholestasis.

Lipopolysaccharides (LPS) are known to cause cholestasis in sepsis. There is evidence that a defective expression of canalicular aquaporin water channels contributes to bile secretory failure in LPS-induced cholestasis. Thus, we studied whether the hepatic adenovirus-mediated transfer of human aquaporin-1 gene (haqp1) can improve the cholestasis induced by LPS. Adenoviral vector encoding hAQP1 (AdhAQP1) or control vector was administered to rats by retrograde intrabiliary infusion. Hepatocyte canalicular hAQP1 expression was assessed by liver immunostaining and immunoblotting in purified plasma membranes. LPS reduced bile flow and biliary bile acid excretion by 30% and 45%, respectively. AdhAQP1-treatment normalized both bile flow and biliary bile acid excretion in LPS-induced cholestasis. Moreover, markedly elevated serum bile acid levels in cholestatic rats, were also normalized with the AdhAQP1 hepatic transduction. Bile flow and serum or biliary bile acids in normal rats were not significantly altered by AdhAQP1. AdhAQP1 delivery unaffected the downregulated protein expression of canalicular bile salt export pump (BSEP/ABCB11) in cholestasis, but improved its transport activity restoring reduced canalicular cholesterol content. Our data suggest that the adenovirus-mediated hepatocyte hAQP1 expression improves LPS-induced cholestasis in rats by stimulating the BSEP/ABCB11-mediated biliary bile acid excretion; a finding that might contribute to the understanding and treatment of sepsis-associated cholestatic diseases. © 2017 IUBMB Life, 69(12):978-984, 2017.

Cholestatic drugs induce preferential accumulation of toxic bile acids in human HepaRG cells

Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50 years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50 mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2 hours (h) after dosing and returned to baseline values before 24 h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.

Editor's Highlight: Clofibrate Decreases Bile Acids in Livers of Male Mice by Increasing Biliary Bile Acid Excretion in a PPARα-Dependent Manner.

Fibrates and their receptor, namely peroxisome proliferator-activated receptor α (PPARα), have been reported to regulate bile acid (BA) synthesis and transport. However, the effect of fibrate treatment and PPARα activation on BA homeostasis remains controversial. In this study, both wild-type (WT) and PPARα-null male mice were treated with clofibrate (CLOF) for 4 days to evaluate the effects of short-term PPARα activation on BA homeostasis. Although a decrease in total BAs (ΣBAs) was observed in livers of CLOF-treated WT mice, it was not observed in PPARα-null mice. CLOF-mediated decrease in ΣBAs in the liver was not likely due to the reduction in BA synthesis or BA uptake, as evidenced by an increase in the BA synthetic enzyme (Cyp7a1) and 2 BA uptake transporters (Na (+)-taurocholate cotransporting polypeptide [Ntcp] and organic anion transporting polypeptide [Oatp]1b2). Instead, the decrease in liver BAs by CLOF is largely a result of increased biliary excretion of BAs, which was associated with a significant induction of the canalicular efflux transporter (bile salt export pump [Bsep]) in the liver. The PPARα-mediated increase in Cyp7a1 in CLOF-treated WT mice was not due to farnesoid X receptor (Fxr)-small heterodimer partner (Shp) signaling in the liver, but due to suppression of Fxr- fibroblast growth factor15 signaling in the ileum. Additionally, CLOF also suppressed intestinal BA transporters (apical sodium-dependent bile acid transporter and organic solute transporterβ) and cholesterol efflux transporters (Abcg5 and Abcg8) in a PPARα-dependent manner. In summary, this study provides the first comprehensive analysis on the effect of a short-term CLOF treatment on BA homeostasis, and revealed an essential role of PPARα in regulating BA synthesis, transport and signaling.

PPAR\u03b1 activation protects against cholestatic liver injury

Intrahepatic cholestasis induced by drug toxicity, bile salt export pump (BSEP) deficiency, or pregnancy frequently causes cholestatic liver damage, which ultimately may lead to liver fibrosis and cirrhosis. Here, the preventive and therapeutic effects of peroxisome proliferator-activated receptor α (PPARα) signaling activated by fenofibrate was evaluated on cholestatic liver damage. Metabolomic analysis revealed that alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis resulted in the accumulation of serum long-chain acylcarnitines and triglyceride, and the reduced expression of four fatty acid β-oxidation (β-FAO) relevant genes (Cpt1b, Cpt2, Mcad and Hadha), indicating the disruption of β-FAO. The increase of acylcarnitines in hepatic cell resulted in the enhanced expression of anti-oxidative genes glutathione S-transferases (Gsta2 and Gstm3) directly. As direct PPARα-regulated genes, Cpt1b, Cpt2, and Mcad were up-regulated after pretreatment with PPARα agonist, fenofibrate, indicating the improvement of β-FAO. In the end, the disrupted bile acid metabolism in the enterohepatic circulation and the enhanced oxidative stress and inflammation cytokines induced by ANIT exposure were significantly recovered with the improvement of β-FAO using fenofibrate treatment. These findings provide the rationale for the use of PPARα agonists as therapeutic alternatives for cholestatic liver damage.

Assessment of the Drug Interaction Potential of Unconjugated and GalNAc3-Conjugated 2'-MOE-ASOs.

Antisense oligonucleotides are metabolized by nucleases and drug interactions with small drug molecules at either the cytochrome P450 (CYP) enzyme or transporter levels have not been observed to date. Herein, a comprehensive in vitro assessment of the drug-drug interaction (DDI) potential was carried out with four 2′-O-(2-methoxyethyl)-modified antisense oligonucleotides (2′-MOE-ASOs), including a single triantennary N-acetyl galactosamine (GalNAc3)-conjugated ASO. Several investigations to describe the DDI potential of a 2′-MOE-ASO conjugated to a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors are explored. The inhibition on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 and induction on CYP1A2, CYP2B6, and CYP3A4 were investigated in cryopreserved hepatocytes using up to 100 μM of each ASO. No significant inhibition (half maximal inhibitory concentration [IC50] > 100 μM) or induction was observed based on either enzymatic phenotype or mRNA levels. In addition, transporter interaction studies were conducted with nine major transporters per recommendations from regulatory guidances and included three hepatic uptake transporters, organic cation transporter 1 (OCT1), organic anion transporting polypeptide 1B1 (OATP1B1), and OATP1B3; three renal uptake transporters, organic anion transporter 1 (OAT1), OAT3, and OCT2; and three efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and bile salt export pump (BSEP). None of the four ASOs (10 μM) were substrates of any of the nine transporters, with uptake <2-fold compared to controls, and efflux ratios were below 2.0 for BCRP and P-gp. Additionally, neither of the four ASOs showed meaningful inhibition on any of the nine transporters tested, with the mean percent inhibition ranging from −38.3% to 24.2% with 100 μM ASO. Based on these findings, the unconjugated and GalNAc3-conjugated 2′-MOE-ASOs would have no or minimal DDI with small drug molecules via any major CYP enzyme or drug transporters at clinically relevant exposures.

Cholestasis After Pediatric Liver Transplantation–Recurrence of a Progressive Familial Intrahepatic Cholestasis Phenotype as a Rare Differential Diagnosis: A Case Report

IntroductionNonobstructive cholestasis after pediatric liver transplantation is a common diagnostic and therapeutic dilemma. We describe a girl with neonatal cholestasis because of progressive familial intrahepatic cholestasis 2 (PFIC-2) and presence of a homozygous splice site mutation in the ABCB11 gene. Liver transplantation was performed because of end-stage liver disease at the age of 6. Cholestasis with normal gamma-glutamyl transferase (GGT) developed 8 years after liver transplantation. A liver biopsy showed canalicular cholestasis and giant cell hepatitis without evidence of rejection, mimicking PFIC-2. Immunofluorescence staining of normal human liver sections with patient's serum revealed reactivity toward a canalicular epitope, which could be identified as bile salt export pump (BSEP) using BSEP–yellow fluorescent protein (YFP) transfected cells. Our patient developed a recurrence of a PFIC-2 phenotype due to production of antibodies against BSEP (alloimmune BSEP disease [AIBD]). Intensification of immunosuppressive therapy as well as antibody treatment with plasmapheresis and Rituximab were initiated, leading to stabilization of the clinical condition and depletion of anti-BSEP antibodies in serum. However, after 1 year liver transplantation was necessary again because of end-stage liver insufficiency. Afterward, immunomodulatory treatment consisted of tacrolimus, mycophenolate mofetil, prednisone, immunoadsorption, and high-dose immunoglobulin therapy (1 g/kg/d). ConclusionCholestasis after liver transplantation may indicate an AIBD with a PFIC-2 phenotype. Besides enhancement of immunosuppressive therapy, an antibody depletion with plasmapheresis, immunoadsorption, immunoglobulins, and B-cell depletion represents a therapeutic option.

Dose-related liver injury of Geniposide associated with the alteration in bile acid synthesis and transportation

Fructus Gardenia (FG), containing the major active constituent Geniposide, is widely used in China for medicinal purposes. Currently, clinical reports of FG toxicity have not been published, however, animal studies have shown FG or Geniposide can cause hepatotoxicity in rats. We investigated Geniposide-induced hepatic injury in male Sprague-Dawley rats after 3-day intragastric administration of 100 mg/kg or 300 mg/kg Geniposide. Changes in hepatic histomorphology, serum liver enzyme, serum and hepatic bile acid profiles, and hepatic bile acid synthesis and transportation gene expression were measured. The 300 mg/kg Geniposide caused liver injury evidenced by pathological changes and increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and γ-glutamytransferase (γ-GT). While liver, but not sera, total bile acids (TBAs) were increased 75% by this dose, dominated by increases in taurine-conjugated bile acids (t-CBAs). The 300 mg/kg Geniposide also down-regulated expression of Farnesoid X receptor (FXR), small heterodimer partner (SHP) and bile salt export pump (BSEP). In conclusion, 300 mg/kg Geniposide can induce liver injury with associated changes in bile acid regulating genes, leading to an accumulation of taurine conjugates in the rat liver. Taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA) as well as tauro-α-muricholic acid (T-α-MCA) are potential markers for Geniposide-induced hepatic damage.

Molecular Mechanism of Taurocholate Transport by the Bile Salt Export Pump, an ABC Transporter Associated with Intrahepatic Cholestasis.

The bile salt export pump (BSEP/ABCB11) transports bile salts from hepatocytes into bile canaliculi. Its malfunction is associated with severe liver disease. One reason for functional impairment of BSEP is systemic administration of drugs, which as a side effect inhibit the transporter. Therefore, drug candidates are routinely screened for potential interaction with this transporter. Hence, understanding the functional biology of BSEP is of key importance. In this study, we engineered the transporter to dissect interdomain communication paths. We introduced mutations in noncanonical and in conserved residues of either of the two nucleotide binding domains and determined the effect on BSEP basal and substrate-stimulated ATPase activity as well as on taurocholate transport. Replacement of the noncanonical methionine residue M584 (Walker B sequence of nucleotide binding site 1) by glutamate imparted hydrolysis competency to this site. Importantly, this mutation was able to sustain 15% of wild-type transport activity, when the catalytic glutamate of the canonical nucleotide binding site 2 was mutated to glutamine. Kinetic modeling of experimental results for the ensuing M584E/E1244Q mutant suggests that a transfer of hydrolytic capacity from the canonical to the noncanonical nucleotide binding site results in loss of active and adoption of facilitative characteristics. This facilitative transport is ATP-gated. To the best of our knowledge, this result is unprecedented in ATP-binding cassette proteins with one noncanonical nucleotide binding site. Our study promotes an understanding of the domain interplay in BSEP as a basis for exploration of drug interactions with this transporter.

Sequencing of FIC1, BSEP and MDR3 in a large cohort of patients with cholestasis revealed a high number of different genetic variants

The bile salt export pump (BSEP, ABCB11), multidrug resistance protein 3 (MDR3, ABCB4) and the ATPase familial intrahepatic cholestasis 1 (FIC1, ATP8B1) mediate bile formation. This study aimed to determine the contribution of mutations and common variants in the FIC1, BSEP and MDR3 genes to cholestatic disorders of differing disease onset and severity. Coding exons with flanking intron regions of ATP8B1, ABCB11, and ABCB4 were sequenced in cholestatic patients with assumed genetic cause. The effects of new variants were evaluated by bioinformatic tools and 3D protein modeling. In 427 patients with suspected inherited cholestasis, 149 patients carried at least one disease-causing mutation in FIC1, BSEP or MDR3, respectively. Overall, 154 different mutations were identified, of which 25 were novel. All 13 novel missense mutations were disease-causing according to bioinformatics analyses and homology modeling. Eighty-two percent of patients with at least one disease-causing mutation in either of the three genes were children. One or more common polymorphism(s) were found in FIC1 in 35.3%, BSEP in 64.3% and MDR3 in 72.6% of patients without disease-causing mutations in the respective gene. Minor allele frequencies of common polymorphisms in BSEP and MDR3 varied in our cohort compared to the general population, as described by gnomAD. However, differences in ethnic background may contribute to this effect. In a large cohort of patients, 154 different variants were detected in FIC1, BSEP, and MDR3, 25 of which were novel. In our cohort, frequencies for risk alleles of BSEP (p.V444A) and MDR3 (p.I237I) polymorphisms were significantly overrepresented in patients without disease-causing mutation in the respective gene, indicating that these common variants can contribute to a cholestatic phenotype. FIC1, BSEP, and MDR3 represent hepatobiliary transport proteins essential for bile formation. Genetic variants in these transporters underlie a broad spectrum of cholestatic liver diseases. To confirm a genetic contribution to the patients' phenotypes, gene sequencing of these three major cholestasis-related genes was performed in 427 patients and revealed 154 different variants of which 25 have not been previously reported in a database. In patients without a disease-causing mutation, common genetic variants were detected in a high number of cases, indicating that these common variants may contribute to cholestasis development.

Rifampicin-induced injury in HepG2 cells is alleviated by TUDCA via increasing bile acid transporters expression and enhancing the Nrf2-mediated adaptive response

Bile acid transporters and the nuclear factor erythroid 2-related factor (Nrf-2)-mediated adaptive response play important roles in the development of drug-induced liver injury (DILI). However, little is known about the contribution of the adaptive response to rifampicin (RFP)-induced cell injury. In this study, we found RFP decreased the survival rate of HepG2 cells and increased the levels of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), γ-glutamyl-transferase (γ-GT), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total bile acid (TBA) and adenosine triphosphate (ATP) in the cell culture supernatants in both a concentration- and a time-dependent manner. RFP increased the expression levels of bile acid transporter proteins and mRNAs, such as bile salt export pump (BSEP), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), Na+/taurocholate cotransporter (NTCP), organic anion transporting protein 2 (OATP2), organic solute transporter β (OSTβ) and Nrf2. Following the transient knockdown of Nrf2 and treatment with RFP, the expression levels of the BSEP, MDR1, MRP2, NTCP, OATP2 and OSTβ proteins and mRNAs were decreased to different degrees. Moreover, the cell survival was decreased, whereas the LDH level in the cell culture supernatant was increased. Overexpression of the Nrf2 gene produced the opposite effects. Treatment with tauroursodeoxycholic acid (TUDCA) increased the expression levels of the bile acid transporters and Nrf2, decreased the expression levels of glucose-regulated protein 78 (GRP78), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP-homologous protein (CHOP), and inhibited RFP-induced oxidative stress. Moreover, TUDCA reduced cell apoptosis, increased cell survival and decreased the levels of LDH, ALT, AST, AKP, γ-GT, TBIL, DBIL, IBIL, TBA and ATP in the cell culture supernatant. Therefore, TUDCA alleviates RFP-induced injury in HepG2 cells by enhancing bile acid transporters expression and the Nrf2-mediated adaptive response.

Bile Salt Homeostasis in Normal and Bsep Gene Knockout Rats with Single and Repeated Doses of Troglitazone.

The interference of bile acid secretion through bile salt export pump (BSEP) inhibition is one of the mechanisms for troglitazone (TGZ)-induced hepatotoxicity. Here, we investigated the impact of single or repeated oral doses of TGZ (200 mg/kg/day, 7 days) on bile acid homoeostasis in wild-type (WT) and Bsep knockout (KO) rats. Following oral doses, plasma exposures of TGZ were not different between WT and KO rats, and were similar on day 1 and day 7. However, plasma exposures of the major metabolite, troglitazone sulfate (TS), in KO rats were 7.6- and 9.3-fold lower than in WT on day 1 and day 7, respectively, due to increased TS biliary excretion. With Bsep KO, the mRNA levels of multidrug resistance-associated protein 2 (Mrp2), Mrp3, Mrp4, Mdr1, breast cancer resistance protein (Bcrp), sodium taurocholate cotransporting polypeptide, small heterodimer partner, and Sult2A1 were significantly altered in KO rats. Following seven daily TGZ treatments, Cyp7A1 was significantly increased in both WT and KO rats. In the vehicle groups, plasma exposures of individual bile acids demonstrated variable changes in KO rats as compared with WT. WT rats dosed with TGZ showed an increase of many bile acid species in plasma on day 1, suggesting the inhibition of Bsep. Conversely, these changes returned to base levels on day 7. In KO rats, alterations of most bile acids were observed after seven doses of TGZ. Collectively, bile acid homeostasis in rats was regulated through bile acid synthesis and transport in response to Bsep deficiency and TGZ inhibition. Additionally, our study is the first to demonstrate that repeated TGZ doses can upregulate Cyp7A1 in rats.

Identification of Bile Acids Responsible for Inhibiting the Bile Salt Export Pump, Leading to Bile Acid Accumulation and Cell Toxicity in Rat Hepatocytes

Inhibition of bile salt export pump (BSEP) causes hepatic accumulation of toxic bile acid (BA), leading to hepatocyte death. We reported a sandwich-cultured hepatocyte (SCH)–based model that can estimate potential cholestatic compounds by assessing their ability to induce hepatotoxicity in combination with a titrated amount of human 12 BA species. However, there is little information about the specific BAs responsible for hepatotoxicity, when BSEP is inhibited. This study measured the accumulation of each BA in rat SCHs in the presence of 10 μM cyclosporine A (CsA), which only inhibits BSEP, and 50 μM CsA, which further inhibits basolateral BA efflux transporters. The accumulation of all BAs (not significant for deoxycholic acid [DCA]) was observed in the presence of 10 μM CsA. In particular, 3 BAs (chenodeoxycholic acid [CDCA], DCA, and glyco-DCA [GDCA]) showed increased toxicity in the presence of 10 μM CsA, whereas the other BAs did not. In addition to these BAs, taurolithocholic acid, glyco-CDCA, and glycocholic acid showed increased toxicity in the presence of 50 μM CsA, but additional accumulation of these BAs could not be observed. These results indicate the inhibiting BSEP results in the accumulation of CDCA, GDCA, and partially DCA, thereby resulting in hepatotoxicity.

Pectin penta-oligogalacturonide reduces cholesterol accumulation by promoting bile acid biosynthesis and excretion in high-cholesterol-fed mice

Haw pectin penta-oligogalacturonide (HPPS) has important role in improving cholesterol metabolism and promoting the conversion of cholesterol to bile acids (BA) in mice fed high-cholesterol diet (HCD). However, the mechanism is not clear. This study aims to investigate the effects of HPPS on cholesterol accumulation and the regulation of hepatic BA synthesis and transport in HCD-fed mice. Results showed that HPPS significantly decreased plasma and hepatic TC levels but increased plasma high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (apoA-I) levels, compared to HCD. BA analysis showed that HPPS markedly decreased hepatic and small intestine BA levels but increased the gallbladder BA levels, and finally decreased the total BA pool size, compared to HCD. Studies of molecular mechanism revealed that HPPS promoted hepatic ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor BI (SR-BI) expression but did not affect ATB binding cassette transporter G5/G8 (ABCG5/8) expression. HPPS inactivated hepatic farnesoid X receptor (FXR) and target genes expression, which resulted in significant increase of cholesterol 7α-hydroxylase 1 (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) expression, with up-regulations of 204.2% and 33.5% for mRNA levels, respectively, compared with HCD. In addition, HPPS markedly enhanced bile salt export pump (BSEP) expression but didn't affect the sodium/taurocholate co-transporting polypeptide (NTCP) expression. In conclusion, the study revealed that HPPS reduced cholesterol accumulation by promoting BA synthesis in the liver and excretion in the feces, and might promote macrophage-to-liver reverse cholesterol transport (RCT) but did not liver-to-fecal RCT.