Tetrahydroxy bile acids (THBAs) are hydrophilic and are present at minimal or undetectable levels in healthy human adults, but are present at high levels in bile salt export pump (abcb11)-knockout mice. The roles of THBAs in human cholestatic diseases are unclear. We aimed to investigate the presence of THBAs in patients with infantile intrahepatic cholestasis and its correlation with outcome. Urinary bile acids (BAs) were analyzed by GC-MS. Data were compared between good (n = 21) (disease-free before 1 year old) and poor prognosis groups (n = 19). Good prognosis patients had a higher urinary THBA proportion than poor prognosis patients [25.89% (3.45-76.73%) vs. 1.93% (0.05-48.90%)]. A urinary THBA proportion >7.23% predicted good prognosis with high sensitivity (95.24%), specificity (84.21%), and area under the curve (0.91) (P < 0.0001). A THBA proportion 7.23% was an independent factor for decreased transplant-free survival (hazard ratio = 7.16, confidence interval: 1.24-41.31, P = 0.028). Patients with a confirmed ABCB11 or tight junction protein 2 gene mutation (n = 7) had a minimally detectable THBA proportion (0.23-2.99% of total BAs). Three patients with an ATP8B1 mutation had an elevated THBA proportion (7.51-37.26%). In conclusion, in addition to disease entity as a major determinant of outcome, a high THBA level was associated with good outcome in the infantile intrahepatic cholestasis patients.

Ursodeoxycholic acid (UDCA), a natural, dihydroxy bile acid, promotes gallstone dissolution and has been attributed with several other beneficial effects. The farnesoid X receptor (FXR) may influence the pharmacokinetics of UDCA by modulating the expression of bile acid transporters. This exploratory study examined whether common functional polymorphisms in FXR and in bile acid transporter genes affect the pharmacokinetics of exogenous UDCA. Polymorphisms in genes for transporters involved in bile acid transport, solute carrier organic anion 1B1 (SLCO1B1) 388A>G and 521T>C, solute carrier 10A1 (SLC10A1) 800 C>T and ATP-binding cassette B11 (ABCB11) 1331T>C, and the FXR -1G>T polymorphism were genotyped in 26 male Chinese subjects who ingested single oral 500-mg doses of UDCA. Plasma concentrations of UDCA and its major conjugate metabolite glycoursodeoxycholic acid (GUDCA) were determined. The mean systemic exposure of UDCA was higher in the five subjects with one copy of the FXR -1G>T variant allele than in those homozygous for the wild-type allele (n = 21) (AUC0-24 h : 38.5 ± 28.2 vs. 20.9 ± 8.0 μg h/mL, P = 0.021), but this difference appeared mainly due to one outlier with the -1GT genotype and elevated baseline and post-treatment UDCA concentrations. After excluding the outlier, body weight was the only factor associated with plasma concentrations of UDCA and there were no significant associations with the other polymorphisms examined. None of the polymorphisms affected the pharmacokinetics of GUDCA. This study showed that the common polymorphisms in bile acid transporters had no significant effect on the pharmacokinetics of exogenous UDCA but an effect of the FXR polymorphism cannot be excluded.

Evidence indicates that the polymorphisms in bile salt export pump (BSEP, encoded by ABCB11) may play an important role in the development of anti-tuberculosis drug-induced liver injury (ATDILI) and we aim to investigate the association between genetic variants of ABCB11 and the risk of ATDILI in a Chinese cohort. A total of 89 tuberculosis patients with ATDILI and 356 matched ATDILI -free patients constituted cases and controls. Genetic polymorphisms of ABCB11 were determined by TaqMan single-nucleotide polymorphism (SNP) genotyping assay. Odds ratio (OR) with 95% confidence intervals (CIs) was estimated by conditional logistic regression model. There were no significant differences in genotype frequencies of ABCB11 between cases and controls. In the subgroup analysis, polymorphisms of rs2287616 were found to be associated with cholestatic/mixed pattern of liver injury under dominant and addictive model (OR = 3.84, 95% CI:1.16-12.75, P = 0.028 and OR = 2.51, 95% CI:1.12-5.62, P = 0.025, respectively), however the significance disappeared after Bonferroni correction. This study suggested that genetic variants of ABCB11 gene might contribute to anti-tuberculosis drug-induced cholestatic liver injury in Chinese patients. Studies in larger, varied populations are required to confirm these findings.

Placenta performs the function of several adult organs for the fetus during intrauterine life. Because of the dramatic physiological and metabolic changes during pregnancy and the strong association between maternal metabolism and placental function, the possibility that variation in gene expression patterns during pregnancy might be linked to fetal health warrants investigation. Here, next-generation RNA sequencing was used to investigate the expression profile, including mRNAs and long non-coding RNAs (lncRNAs) of placentas on day 60 of gestation (G60), day 90 of gestation (G90), and on the farrowing day (L0) in pregnant swine. Bioinformatics analysis of differentially expressed mRNAs and lncRNAs consistently showed dysregulation of bile acids transport and detoxification as pregnancy progress. We found the differentially expressed mRNAs, particularly bile salt export pump (ABCB11), organic anion-transporting polypeptide 1A2 (OATP1A2), carbonic anhydrase II (CA2), Na⁺-HCO₃^- cotransporter (NBC1), and hydroxysteroid sulfotransferases (SULT2A1) play an important role in bile acids transport and sulfation in placentas during pregnancy. We also found the potential regulation role of ALDBSSCG0000000220 and XLOC_1301271 on placental SULT2A1. These findings have uncovered a previously unclear function and its genetic basis for bile acids metabolism in developing placentas and have important implications for exploring the potential physiological and pathological pathway to improve fetal outcomes.

In the present study, we conducted an RNA-Seq analysis to characterize the genes and pathways involved in acute thermal and cold stress responses in the liver of black rockfish, a viviparous teleost that has the ability to cope with a wide range of temperature changes. A total of 584 annotated differentially expressed genes (DEGs) were identified in all three comparisons (HT vs NT, HT vs LT and LT vs NT). Based on an enrichment analysis, DEGs with a potential role in stress accommodation were classified into several categories, including protein folding, metabolism, immune response, signal transduction, molecule transport, membrane, and cell proliferation/apoptosis. Considering that thermal stress has a greater effect than cold stress in black rockfish, 24 shared DEGs in the intersection of the HT vs LT and HT vs NT groups were enriched in 2 oxidation-related gene ontology (GO) terms. Nine important heat-stress-reducing pathways were significantly identified and classified into 3 classes: immune and infectious diseases, organismal immune system and endocrine system. Eight DEGs (early growth response protein 1, bile salt export pump, abcb11, hsp70a, rtp3, 1,25-dihydroxyvitamin d(3) 24-hydroxylase, apoa4, transcription factor jun-b-like and an uncharacterized gene) were observed among all three comparisons, strongly implying their potentially important roles in temperature stress responses.

BACKGROUND

Primary human hepatocytes are a useful in vitro model system to examine hepatic biochemical pathways, liver disorders and/or pharmacotherapies. This system can also be used for transport studies to investigate uptake and excretion of bile acids. Proper modeling of hepatic function requires careful attention to media components, and culture substrates and conditions.

OBJECTIVE

To examine the effects of different culture media and conditions on bile acid transport in cultured human hepatocytes.

RESULTS

Hepatocytes cultured in Williams' medium E showed an increase in both uptake and excretion of taurocholate compared to cells cultured in Dulbecco's Modified Eagle Medium (DMEM). Supplementation of DMEM with glutathione or ascorbic acid did not compensate for the lower transport. The difference can be explained by lower mRNA expression of the transporter proteins sodium taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP; ABCB11) when cultured in DMEM. Hepatocytes cultured in DMEM also display fewer and smaller bile canaliculi. Following extended time in culture supplementation of Williams' medium E with dexamethasone increased the expression of NTCP and BSEP.

CONCLUSIONS

Williams' medium E is superior to DMEM for transport studies in primary human hepatocytes. Supplementation with dexamethasone increase mRNA levels of NTCP and BSEP.

Cholesterol cholelithiasis is one of the most common gastroenterological diseases in Western countries. It is a polygenic disease resulting from disturbed biliary cholesterol homeostasis. Association studies identified six human gallstone candidate genes. Polymorphisms in the genes encoding the apolipoproteins B and E, phospholipid flippase ( ABCB4), cholesterol ester transfer protein ( CETP), cholesterol-7alpha-hydroxylase ( CYP7A1) and ileal bile acid transporter ( SLC10A2) are correlated with gallstone prevalence. Quantitative Trait Locus (QTL) analysis localises additional unknown gallstone genes in inbred mice. Based on the natural variation of cholesterol gallstone susceptibility among different inbred strains, 5 lithogenic ( Lith) loci have been identified. Hepatobiliary transporters (e. g. bile salt export pump Abcb11) and key proteins of the lipoprotein metabolism (e. g. hepatic lipase Lipc) could be established as creedal candidate genes for Lith loci. The rapid progress of mouse and human genome projects provides the basis for the analysis of orthologous human LITH genes in gallstone patients, which might offer new prospects for individual risk assessment and molecular targets for stone prevention.

Background & aims: Alterations in the serum levels of bile acids are associated with drug-induced liver injury (DILI). We investigated the association between serum levels of bile acids and the severity and outcome of DILI, along with the potential role of variants in the ATP binding cassette subfamily B member 11 (ABCB11) gene and expression of its product, ABCB11 (also called BSEP).

Methods: We performed this prospective study of 95 patients (median age, 53 years; 73.7% female) with DILI from August 2018 through August 2019. Patients were matched for age, gender, and body mass index with healthy individuals (n=100; healthy controls) and patients with chronic hepatitis B (n=105; CHB controls). We collected demographic and biochemical data at baseline and 1 week, 1 month, 3 months, and 6 months after DILI onset and at the time of biochemical recovery, liver failure or liver transplantation. Serum levels of bile acids were measured using high-performance liquid-chromatography tandem mass-spectrometry. All 27 exons of ABCB11 were sequenced and expression of BSEP were analyzed by immunohistochemistry in liver biopsy specimens.

Results: Levels of 30 of the 37 bile acids analyzed differed significantly between patients with DILI and healthy controls. Changes in levels of taurocholic acid (TCA), glycocholic acid, taurochenodeoxycholate, and glycochenodeoxycholate associated with the increased levels of bilirubin and greater severity of DILI, and were also associated with CHB. Cox regression analysis showed that only change in the levels of TCA independently associated with biochemical resolution of DILI. Combination of TCA level (≥ 1955.41 nmol/L), patient age, and DILI severity was associated with abnormal blood biochemistry at 6 months after DILI onset (area under the curve, 0.81; 95% confidence interval, 0.71-0.88; sensitivity, 0.69; specificity, 0.81). ABCB11 missense variants were not associated with differences in the serum bile acid profiles, DILI severity, or clinical resolution. However, lower levels of BSEP in bile canaliculi in liver biopsies were associated with altered serum levels of bile acids.

Conclusions: In this prospective study performed in Chinese patients, we found that the serum levels of TCA were associated with the severity and clinical resolution of DILI. Reduced protein expression of BSEP in liver tissue, rather than variants of the ABCB11 gene were associated with altered serum levels of bile acids.

Keywords: bile salt export pump; hepatitis B virus; herbal supplement; toxicity.

The etiology of intrahepatic cholestasis of pregnancy (ICP) involves environmental, hormonal and genetic factors. It is thought that ICP may be related to the polymorphic variants of several genes involved in the metabolism and transport of bile acids (BA). The goal of our study was to evaluate the possible role of genetic polymorphic variants of ABC transporters in patients with ICP.

96 women with ICP (mean age of 30.42 years, mean gestational age of 36.83 gestation weeks) and 211 healthy pregnant women (mean age of 30.68 years, mean gestational age of 39.05 gestation weeks) were enrolled in the study. Genetic analysis was performed using a polymerase chain reaction / restriction fragment length polymorphism (PCR/RFLP) method. The following polymorphisms were analysed: 1331T>> C (V444A) ABCB11 and 1954A>> G (R652G) ABCB4.

Our analysis of frequency of genotypes and alleles of the 1954A>> G ABCB4 polymorphism revealed no significant differences between the ICP and control groups. For the 1331T>> C polymorphism of the ABCB11 gene the results revealed a higher frequency of 1331CC genotypes in the ICP group (39.58% vs. 29.38%. OR = 1.57, p = 0.05). Also, the frequency of the 1331C allele was higher in the ICP group compared to the control group (64.06% vs. 55.69%, OR = 1.42, p = 0.03).

The overrepresentation of mutated variants of the 1331T>> C ABCB11 polymorphism in the ICP group suggests its contribution to the etiology of the intrahepatic cholestasis of pregnancy. Analysis of genotypes' co-existence pointed to the possibility of the mutated variants of polymorphism 1954A>> G ABCB4 and 1331T>> C ABCB11 having a summation effect on the development of ICP.

Progressive familial intrahepatic cholestasis type 1 is a rare disease that is characterized by low serum γ-glutamyltransferase levels due to mutation in ATP8B1. We present a 23-year-old male who experienced persistent marked pruritus for eighteen years and recurrent jaundice for thirteen years, in addition to cholestasis that eventually became fatal. Genetic sequencing studies of the entire coding (exon) sequences of ATP8B1 and ABCB11 uncovered a novel heterozygous missense 3035G>T mutation (S1012I) and a synonymous 696T>C mutation in ATP8B1. The patient's progression was associated with not only impaired familial intrahepatic cholestasis 1 (FIC1) function but also impaired bile salt export pump expression due to the impaired FIC1 function. Our findings show that patients with intermittent cholestasis can develop progressive liver disease even after several decades and require regular follow up.

Cholestasis is characterized by intrahepatic accumulation of potentially cytotoxic bile acids (BAs) subsequently leading to liver injury with disruption of hepatocellular integrity, inflammation, fibrosis and ultimately liver cirrhosis. Bile salt export pump (BSEP/ABCB11) is the main canalicular BA transporter and therefore the rate limiting step for hepatobiliary BA excretion. In this study we aimed to investigate the role of BSEP/ABCB11 in the development of acquired cholestatic liver and bile duct injury.

Wild-type (WT) and BSEP knockout (BSEP-/-) mice were subjected to common bile duct ligation (CBDL) or 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) feeding as models for cholestasis with biliary obstruction and bile duct injury. mRNA expression profile, serum biochemistry, liver histology, immunohistochemistry, hepatic hydroxyproline levels and BA composition as well as biliary pressure were assessed.

BSEP-/- mice were protected against acquired cholestatic liver injury induced by 7days of CBDL or 4weeks of DDC feeding, as reflected by unchanged serum levels of liver transaminases, alkaline phosphatase and BAs. Notably, BSEP-/- mice were also protected from cholestasis-induced hepatic inflammation and biliary fibrosis. In line with induced BA detoxification/hydroxylation pathways in BSEP-/- mice, polyhydroxylated BAs were increased 4-fold after CBDL and 6-fold after DDC feeding in comparison with cholestatic WT mice. Finally, following CBDL, biliary pressure in WT mice increased up to 47mmH2O but remained below 11mmH2O in BSEP-/- mice.

Metabolic preconditioning with subsequent changes in BA metabolism favors detoxification of potentially toxic BAs and thereby protects BSEP-/- mice from cholestatic liver and bile duct injury.

Reduced hepatobiliary bile acid transport due to loss of BSEP function leads to increased hydroxylation of bile acids in the liver. Metabolic preconditioning with a hydrophilic bile pool protects the BSEP-/- mice from acquired cholestatic liver disease.

OBJECTIVE

We explored the role of SNPs within the SLCO1B3, SLCO1B1, SLC22A6, ABCB1, ABCG2, SLCO3A1, CYP2C19, ABCC2, SLC22A1, ABCB11 and NR1I2 genes on voriconazole pharmacokinetics.

METHODS

233 pediatric patients were enrolled. Drug plasma Ctrough was measured by a HPLC-MS method. Allelic discrimination was performed by qualitative real-time PCR.

RESULTS

SLCO1B3 rs4149117 c.334 GT/TT (p = 0.046), ABCG2 rs13120400 c.1194 + 928 CC (p = 0.029) and ABCC2 rs717620 c.-24 GA/AA (p = 0.025) genotype groups significantly influenced Ctrough. ethnicity (p = 0.042), sex (p = 0.033), SLCO1B3 rs4149117 c.334 GT/TT (p = 0.041) and ABCB1 rs1045642 c.3435 TT (p = 0.016) have been retained in linear regression model as voriconazole predictor factors.

CONCLUSIONS

Understanding how some gene polymorphisms affect the voriconazole pharmacokinetic is essential to optimally dose this agent.

Chronic hepatitis C (CHC) is a leading cause of liver disease. Despite the improved efficacy of new antivirals, their high costs preclude their adoption in resource-limited settings, where CHC prevalence is highest. We developed a triplex high-resolution melting assay for the simultaneous assessment of three genetic polymorphisms related to the response to treatment and development of advanced fibrosis in CHC: IFNL3 rs12979860, ABCB11 rs2287622, and RNF7 rs16851720. We validated the assay in clinical samples from 130 CHC patients treated with classic therapy. The assay showed excellent reproducibility and 100% accuracy, sensitivity, and specificity against the gold standard Sanger sequencing. When added to routine examination data, genotype information significantly improved their performance for prediction of advanced liver fibrosis and sustained virological response (P = 0.041 and P = 0.011, respectively). Correspondingly, the full models had area under the receiver operating characteristic curve values of 0.842 (95% CI, 0.773-0.911) and 0.921 (95% CI, 0.870-0.972) and integrated discrimination improvements of 7.5% (95% CI, 2.5%-12.5%; P = 0.003) and 11.5% (95% CI, 5.8%-17.2%; P < 0.001), respectively. This is the first report on a diagnostic test for simultaneous genotyping of IFNL3, ABCB11, and RNF7 in CHC patients. Reliable and inexpensive, the assay should provide useful information for the clinical management of CHC, like identification of patients at risk of rapid disease progression or with high chances of response to classic therapy.

Increased serum bile salt levels have been associated to a single-nucleotide polymorphism in the bile salt export pump (BSEP; ABCB11) in several acquired cholestatic liver diseases but there is little evidence in alcoholic liver disease (ALD). Furthermore, a crosstalk between vitamin D and bile acid synthesis has recently been discovered. Whether this crosstalk has an influence on the course of ALD is unclear to date. Our aim was to analyse the role of genetic polymorphisms in BSEP and the vitamin D receptor gene (NR1I1) on the emergence of cirrhosis in patients with ALD. Therefore, 511 alcoholic patients (131 with cirrhosis and 380 without cirrhosis) underwent ABCB11 genotyping (rs2287622). Of these, 321 (131 with cirrhosis and 190 without cirrhosis) were also tested for NR1I1 polymorphisms (bat-haplotype: BsmI rs1544410, ApaI rs7975232 and TaqI rs731236). Frequencies of ABCB11 and NR1I1 genotypes and haplotypes were compared between alcoholic patients with and without cirrhosis and correlated to serum bile salt, bilirubin and aspartate aminotransferase levels in those with cirrhosis. Frequencies of ABCB11 and NR1I1 genotypes and haplotypes did not differ between the two subgroups and no significant association between genotypes/haplotypes and liver function tests could be determined for neither polymorphism. We conclude that ABCB11 and NR1I1 polymorphisms are obviously not associated with development of cirrhosis in patients with ALD.

Altered expression of drug transporters and metabolic enzymes is known to occur in infection-induced inflammation. We hypothesize that in human immunodeficiency virus (HIV)-infected individuals, further alteration could occur as a result of augmented inflammation. The HIV-1 transgenic (Tg) rat is used to simulate HIV pathologies associated with the presence of HIV viral proteins. Therefore, the objective of this study was to examine the effect of endotoxin administration on the gene expression of drug transporters in the liver of HIV-Tg rats. Male and female HIV-Tg and wild-type (WT) littermates were injected with 5 mg/kg endotoxin or saline (n= 7-9/group). Eighteen hours later, rats were euthanized and tissues were collected. Quantitative real-time polymerase chain reaction and Western blot analysis were used to measure hepatic gene and protein expression, respectively, and enzyme-linked immunosorbent assay was used to measure serum cytokine levels. Although an augmented inflammatory response was seen in HIV-Tg rats, similar endotoxin- mediated downregulation of Abcb1a, Abcc2, Abcg2, Abcb11, Slco1a1, Slco1a2, Slco1b2, Slc10a1, Slc22a1, Cyp3a2, and Cyp3a9 gene expression was seen in the HIV-Tg and WT groups. A significantly greater endotoxin- mediated downregulation of Ent1/Slc29a1 was seen in female HIV-Tg rats. Basal expression of inflammatory mediators was not altered in the HIV-Tg rat; likewise, the basal expression of most transporters was not significantly different between HIV-Tg and WT rats. Our findings suggest that hepatobiliary clearances of endogenous and exogenous substrates are altered in the HIV-Tg rat after endotoxin exposure. This is of particular importance because HIV-infected individuals frequently present with bacterial or viral infections, which are a potential source for drug-disease interactions.

Mithramycin demonstrates preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with germline variants in genes involved in bile disposition: ABCB4 (multidrug resistance 3) rs2302387 and ABCB11 [bile salt export pump (BSEP)] rs4668115 reduce transporter expression (P < 0.05) and were associated with ≥grade 3 transaminitis developing 24 hours after the third infusion of mithramycin (25 mcg/kg, 6 hours/infusion, every day ×7, every 28 days; P < 0.0040). A similar relationship was observed in a pediatric cohort. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, sodium/taurocholate cotransporting polypeptide, organic solute transporter α/β) in several cell lines [Huh7, HepaRG, HepaRG BSEP (-/-)] and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P < 0.0001), and mithramycin inhibited chenodeoxycholic acid- and GW4046-induced FXR-galactose-induced gene 4 luciferase reporter activity (P < 0.001). Mithramycin promoted glycochenodeoxycholic acid-induced cytotoxicity in ABCB11 (-/-) cells and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P < 0.01). Mithramycin is a FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low ABCB11 function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses. SIGNIFICANCE STATEMENT: The present study characterizes a novel mechanism of drug-induced hepatotoxicity in which mithramycin not only alters farnesoid X receptor (FXR) and small heterodimer partner gene expression but also inhibits bile acid binding to FXR, resulting in deregulation of cellular bile homeostasis. Two novel single-nucleotide polymorphisms in bile flow transporters are associated with mithramycin-induced liver function test elevations, and the present results are the rationale for a genotype-directed clinical trial using mithramycin in patients with thoracic malignancies.

OBJECTIVE

Beraprost sodium (BPS) is a chemically stable and orally active prostacyclin analog that is used in the treatment of chronic arterial occlusive disease since 1992 and primary pulmonary hypertension since 1999 in Japan. Multiple-drug therapy is common in clinical practice, and BPS is co-administered with other drugs. Membrane transporters are known to markedly affect pharmacokinetics, safety and efficacy, and many transporter-based drug-drug interactions have been recently reported. However, information on the transporters involved in the pharmacokinetics of BPS is limited.

METHODS

First of all, we have examined 11 transporters, ABCB1 (P-glycoprotein: P-gp), ABCG2 (breast cancer resistance protein: BCRP), SLC22A6 (organic anion transporter 1: OAT1), SLC22A8 (organic anion transporter 3: OAT3), SLCO1B1 (organic anion transporting polypeptide 1B1: OATP1B1), SLCO1B3 (organic anion transporting polypeptide 1B3: OATP1B3), SLCO2B1 (organic anion transporting polypeptide 2B1: OATP2B1), SLC22A1 (organic cation transporter 1: OCT1), SLC22A2 (organic cation transporter 2: OCT2), ABCB11 (bile-salt export pump: BSEP), and ABCC2 (multidrug resistance associated protein 2: MRP2) to clarify which of them would be candidates that might recognize BPS as their substrate in transporter-expressing LLC-PK1, S2, and HEK293 cells as well as in membrane vesicles. Furthermore, we determined whether the transport of BPS was inhibited by the typical inhibitors of each transporter, i.e., verapamil for P-gp, Ko143 for BCRP, probenecid for OAT3, rifampicin for OATP1B1 and OATP1B3, cyclosporine for BSEP, and sulfobromophthalein (BSP) for MRP2.

RESULTS

The results obtained showed that P-gp, BCRP, OAT3, OATP1B1, OATP1B3, BSEP and MRP2 might be candidates for BPS transporters. From the further evaluation with the typical inhibitors of each transporter, it was confirmed that BPS is a substrate for P-gp, BCRP, OAT3, OATP1B1, OATP1B3 and MRP2, because the typical inhibitor, cyclosporine, had no effects on BPS transport by BSEP.

CONCLUSIONS

BPS is a substrate of 6 transporters: P-gp, BCRP, OAT3, OATP1B1, OATP1B3, and MRP2, because their expressing cells and vesicles transported BPS more than in the controls, and BPS transport activities were reduced by the typical inhibitors of tested transporters. Although there are no reports regarding drug-drug interactions between BPS and possible combination drugs expected due to transporters, it may be necessary to notice that that substrates or inhibitors for the 6 mentioned transporters may have effects on pharmacokinetics of BPS when co-administered.

Cholestasis with normal gamma glutamyl transferase characterizes functional deficiencies in the gene ABCB11, which encodes the bile salt export pump (BSEP), a liver-specific adenosine triphosphate (ATP)-binding cassette transporter. Here we report the case of a patient presenting with features of benign recurrent intrahepatic cholestasis associated with a heterozygous mutation in the ABCB11 gene. Immunohistochemistry showed a gradual decrease of BSEP from zone 1 to zone 3 of the liver lobule, suggesting that the mutation identified here may predispose patients to cholestasis through a delocalization process of BSEP at the lobular level. (HEPATOLOGY 2013;57:2539-2541).

Identification of the transport systems involved in bile secretion and of the genes codifying these systems has allowed the etiology of familial intrahepatic cholestasis to be determined in most affected children. Mutations in ATP8B1 cause a defect in FIC1, an aminophospholipid flipase, and give rise to a variable spectrum of disease, ranging from progressive intrahepatic cholestasis to benign recurrent cholestasis, due to alterations in the lipid composition of the membranes and decreased expression of the nuclear factor FXR. Mutations in ABCB11 cause a defect of the canalicular bile salt export pump (BSEP), with early clinical manifestations and progression to hepatocellular failure in childhood. Mutations in ABCB4 cause an alteration in the MDR3 phospholipid transporter, and a variable spectrum of disease from progressive ductal injury to cirrhosis in children, and gallstones, cholestasis of pregnancy, or late cirrhosis in adults.

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, 100 nM), 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, 100 nM), 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.

The identification of biliary tranporters has enhanced our understanding of bile formation and some liver diseases. In this review, we first describe the main hepatobiliary transporters and their function. Then, some liver diseases related to mutations of biliary tranporters (FIC1/ATP8B1, BSEP/ABCB11, MDR3 /ABCB4 and MRP2/ABCC2) will be described with a focus on the pathological aspects. These diseases include progressive familial intrahepatic cholestasis (PFIC), benign recurrent intrahepatic cholestasis (BRIC), intrahepatic cholestasis of pregnancy, Dubin-Johnson's syndrome and low phospholipid associated cholelithiasis (LPAC).

The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We previously reported that 4-phenylbutyrate (4PBA), an approved drug for urea cycle disorders, is a promising agent for intrahepatic cholestasis because it increases both the cell surface expression and the transport capacity of BSEP. In the present study, we searched for effective compounds other than 4PBA by focusing on short- and medium-chain fatty acids, which have similar characteristics to 4PBA such as their low-molecular-weight and a carboxyl group. In transcellular transport studies using Madin-Darby canine kidney (MDCK) II cells, all short- and medium-chain fatty acids tested except for formate, acetate, and hexanoic acid showed more potent effects on wild type (WT) BSEP-mediated [3H]taurocholate transport than did 4PBA. The increase in WT BSEP transport with butyrate and octanoic acid treatment correlated with an increase in its expression at the cell surface. Two PFIC2-type variants, E297G and D482G BSEP, were similarly affected with both compounds treatment. The prolonged half-life of cell surface-resident WT BSEP was responsible for this increased octanoic acid-stimulated transport, but not for that of butyrate. In conclusion, short- and medium-chain fatty acids have potent effects on the increase in WT and PFIC2-type BSEP-mediated transport in MDCK II cells. Although both short- and medium-chain fatty acids enhance the transport capacity of WT and PFIC2-type BSEP by inducing those expressions at the cell surface, the underlying mechanism seems to differ between fatty acids.