Green tea extract was tested for the secondary prevention of colorectal adenoma in the placebo-controlled MIRACLE trial. Genome-wide screening on adenoma recurrence was performed in n = 550 participants 3 years after randomization to green tea or placebo intake.

Single Marker Analysis followed by regression analyses was calculated for all 700.078 markers assuming an additive genetic model and including all covariates from the main MIRACLE trial analysis. The outcome was an adenoma rate at 3-year follow-up colonoscopy comparing participants carrying a genetic variant versus wildtype.

The gene showing the strongest association with the outcome in both, SMA as well as regression analysis, was the organic anion transporter SLCO1A2. In the variant carriers, the adenoma frequency was 41.4% in the green tea group and 35.7% in the placebo group (RR 1.16 [0.81; 1.65] p = 0.61), whereas in the nonvariant carriers, the frequency of reoccurrence was 54.5% in the green tea group and 66.5% in the placebo group (RR 0.82 [0.69; 0.97], p = 0.03).

Individuals with genetic variants in the transporter SLCO1A2 may be protected against colon adenoma irrespective of the green tea intake. In nonvariant carriers of SLCO1A2, green tea was associated with a clear benefit in outcome (18% risk reduction).

We quantified hepatic functional impairment using quantitative function tests and linked severity of functional impairment to liver-related complications and outcome in primary sclerosing cholangitis.

Forty-seven patients had baseline testing, and 40 were retested after 1 year. For each test, cholates labeled with cold, nonradioactive isotopes were administered orally (DuO, SHUNT tests) and intravenously (SHUNT test), and blood was analyzed at 20 and 60 minutes (DuO), or 0, 5, 20, 45, 60, and 90 minutes (SHUNT). Disease severity index (DSI), hepatic reserve (HR%), and portal-systemic shunting (SHUNT%) were calculated.

Three subgroups with low, moderate, and high disease severity were defined from the age-adjusted results for DSI, HR%, and SHUNT%. Standard laboratory tests, clinical scores, cytokine levels, and clinical outcome correlated with these subgroups. In univariate analysis of baseline tests, SHUNT% was a strong predictor of clinical outcome (n = 13 of 47; areas under the receiver operating characteristic curve, 0.84DuO, 0.90SHUNT). A model combining SHUNT%, DSI (or HR%), platelet count, and changes from baseline was most predictive of outcome (n = 10 of 40; areas under the receiver operating characteristic curve, 0.95DuO, 0.96SHUNT).

DSI, HR%, and SHUNT% identified subgroups of primary sclerosing cholangitis based on the age-related severity of hepatic impairment that predicted risk for liver-related clinical outcome. Further study is warranted to confirm and validate these intriguing findings both in studies of natural progression of primary sclerosing cholangitis and in clinical trials. DuO enhances the utility of quantitative liver function testing.

Bile acids (BA) play an important role in cholesterol metabolism and possess further beneficial metabolic effects as signalling molecules. Blocking the hepatocellular uptake of BA via sodium-taurocholate co-transporting polypeptide (NTCP) with the first-in-class drug bulevirtide, we expected to observe a decrease in plasma LDL cholesterol. In this exploratory phase I clinical trial, volunteers with LDL cholesterol > 130 mg/dL but without overt atherosclerotic disease were included. Thirteen participants received bulevirtide 5 mg/d subcutaneously for 12 weeks. The primary aim was to estimate the change in LDL cholesterol after 12 weeks. Secondary endpoints included changes in total cholesterol, HDL cholesterol, lipoprotein(a), inflammatory biomarkers, and glucose after 12 weeks. In addition, cardiac magnetic resonance imaging (CMR) was performed at four time points. BA were measured as biomarkers of the inhibition of hepatocellular uptake. After 12 weeks, LDL cholesterol decreased not statistically significantly by 19.6 mg/dL [−41.8; 2.85] (Hodges−Lehmann estimator with 95% confidence interval). HDL cholesterol showed a significant increase by 5.5 mg/dL [1.00; 10.50]. Lipoprotein(a) decreased by 1.87 mg/dL [−7.65; 0]. Inflammatory biomarkers, glucose, and cardiac function were unchanged. Pre-dose total BA increased nearly five-fold (from 2026 nmol/L ± 2158 (mean ± SD) at baseline to 9922 nmol/L ± 7357 after 12 weeks of treatment). Bulevirtide was generally well tolerated, with most adverse events being administration site reactions. The exploratory nature of the trial with a limited number of participants allows the estimation of potential effects, which are crucial for future pharmacological research on bile acid metabolism in humans.

Drug-drug interactions mediated by transporters are a serious clinical concern hence a tremendous amount of work has been done on the characterization of the transporter-mediated proteins in humans and animals. The underlying mechanism for the transporter-mediated drug-drug interaction is the induction or inhibition of the transporter which is involved in the cellular uptake and efflux of drugs. Transporter of the brain, liver, kidney, and intestine are major determinants that alter the absorption, distribution, metabolism, excretion profile of drugs, and considerably influence the pharmacokinetic profile of drugs. As a consequence, transporter proteins may affect the therapeutic activity and safety of drugs. However, mounting evidence suggests that many drugs change the activity and/or expression of the transporter protein. Accordingly, evaluation of drug interaction during the drug development process is an integral part of risk assessment and regulatory requirements. Therefore, this review will highlight the clinical significance of the transporter, their role in disease, possible cause underlying the drug-drug interactions using analytical tools, and update on the regulatory requirement. The recent in-silico approaches which emphasize the advancement in the discovery of drug-drug interactions are also highlighted in this review. Besides, we discuss several endogenous biomarkers that have shown to act as substrates for many transporters, which could be potent determinants to find the drug-drug interactions mediated by transporters. Transporter-mediated drug-drug interactions are taken into consideration in the drug approval process therefore we also provided the extrapolated decision trees from in-vitro to in-vivo, which may trigger the follow-up to clinical studies.

Organic anion transporting polypeptides (OATPs; gene symbol SLCO) are membrane transporters that mediate the transport of wide ranges of compounds. The expression of different OATP members has been reported in the kidney, liver, placenta, brain, and intestine. Because of their broad substrate spectra and wide distribution within the human body, these transporters have been proposed to play key roles in the influx transport of many oral drugs. Inflammation is known to regulate the expression and functions of many drug-metabolizing enzymes and drug transporters. As a proinflammatory cytokine, tumor necrosis factor-α (TNFα) has been shown to affect the expression of different drug transporters, including OATP family members. In the present study, a putative nuclear factor-κB (NFκB) binding site ranging from -1845 to -1836 was identified at the proximal promoter region of OATP1A2 coding gene SLCO1A2 Electrophoretic mobility shift assays and chromatin immunoprecipitation showed that nuclear extracts from both breast cancer cell MCF7 and liver cancer cell HepG2 interacted with an oligonucleotide probe containing the putative NFκB binding site and that the DNA-protein complexes contained both p65 and p50 subunits of NFκB. Further study revealed that the binding site may be responsible in part for the suppression effect of TNFα toward SLCO1A2 expression because the treatment of TNFα significantly increased. Treatment of TNFα significantly increased formation of the DNA-protein complexes and mutations at essential bases of the putative NFκB binding site abolished responsiveness to the TNFα neutralizing antibody, suggesting that the binding site may be responsible in part for the suppression effect of TNFα towars SLCO1A2 expression.

Organic anion transporters (OATs) and organic anion-transporting polypeptides (OATPs), encoded by a number of solute carrier (SLC)22A and SLC organic anion (SLCO) genes, mediate the absorption and distribution of drugs and other xenobiotics. The regulation of OATs and OATPs is complex, comprising both transcriptional and post-translational mechanisms. Plasma membrane expression is required for cellular substrate influx by OATs/OATPs. Thus, interest in post-translational regulatory processes, including membrane targeting, endocytosis, recycling and degradation of transporter proteins, is increasing because these are critical for plasma membrane expression. After being synthesized, transporters undergo N-glycosylation in the endoplasmic reticulum and Golgi apparatus and are delivered to the plasma membrane by vesicular transport. Their expression at the cell surface is maintained by de novo synthesis and recycling, which occurs after clathrin- and/or caveolin-dependent endocytosis of existing protein. Several studies have shown that phosphorylation by signalling kinases is important for the internalization and recycling processes, although the transporter protein does not appear to be directly phosphorylated. After internalization, transporters that are targeted for degradation undergo ubiquitination, most likely on intracellular loop residues. Epigenetic mechanisms, including methylation of gene regulatory regions and transcription from alternate promoters, are also significant in the regulation of certain SLC22A/SLCO genes. The membrane expression of OATs/OATPs is dysregulated in disease, which affects drug efficacy and detoxification. Several transporters are expressed in the cytoplasmic subcompartment in disease states, which suggests that membrane targeting/internalization/recycling may be impaired. This article focuses on recent developments in OAT and OATP regulation, their dysregulation in disease and the significance for drug therapy.

Steroid sulfation and desulfation are fundamental pathways vital for a functional vertebrate endocrine system. After biosynthesis, hydrophobic steroids are sulfated to expedite circulatory transit. Target cells express transmembrane organic anion-transporting polypeptides that facilitate cellular uptake of sulfated steroids. Once intracellular, sulfatases hydrolyze these steroid sulfate esters to their unconjugated, and usually active, forms. Because most steroids can be sulfated, including cholesterol, pregnenolone, dehydroepiandrosterone, and estrone, understanding the function, tissue distribution, and regulation of sulfation and desulfation processes provides significant insights into normal endocrine function. Not surprisingly, dysregulation of these pathways is associated with numerous pathologies, including steroid-dependent cancers, polycystic ovary syndrome, and X-linked ichthyosis. Here we provide a comprehensive examination of our current knowledge of endocrine-related sulfation and desulfation pathways. We describe the interplay between sulfatases and sulfotransferases, showing how their expression and regulation influences steroid action. Furthermore, we address the role that organic anion-transporting polypeptides play in regulating intracellular steroid concentrations and how their expression patterns influence many pathologies, especially cancer. Finally, the recent advances in pharmacologically targeting steroidogenic pathways will be examined.

The organic anion transporting polypeptide 1A2 (OATP1A2), a membrane drug transporter expressed on important organs (such as the brain, kidney, and intestine) may be a key element in the disposition of drugs. Previous studies demonstrated that it could transport a broad spectrum of substrates, including endogenous molecules and clinically relevant drugs, such as several β-blockers and 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors. The primary objective of this study was to investigate OATP1A2 transport activity using rosuvastatin as a probe substrate and evaluate competitive inhibition of its transport by β-blockers. Rosuvastatin transport was saturable, with a Km of 60.2 µM. With the exception of carvedilol (IC50 of 3.2 µM), all of the other β-blockers that were evaluated had a small or insignificant effect on OATP1A2-mediated uptake of rosuvastatin. Carvedilol differs from the other β-blockers by the tricyclic moiety in its chemical structure. As a secondary objective, the transport of a series of tricyclic compounds by OATP1A2 and their potential for rosuvastatin transport inhibition were evaluated. Tricyclic compounds were not OATP1A2 substrates. On the other hand, tricyclic compounds with a short aliphatic amine chain inhibited OATP1A2-mediated rosuvastatin transport. Our data suggest that these drugs may modulate the transport of OATP1A2 substrates and may affect drug actions.

The human organic anion and cation transporters are classified within two SLC superfamilies. Superfamily SLCO (formerly SLC21A) consists of organic anion transporting polypeptides (OATPs), while the organic anion transporters (OATs) and the organic cation transporters (OCTs) are classified in the SLC22A superfamily. Individual members of each superfamily are expressed in essentially every epithelium throughout the body, where they play a significant role in drug absorption, distribution and elimination. Substrates of OATPs are mainly large hydrophobic organic anions, while OATs transport smaller and more hydrophilic organic anions and OCTs transport organic cations. In addition to endogenous substrates, such as steroids, hormones and neurotransmitters, numerous drugs and other xenobiotics are transported by these proteins, including statins, antivirals, antibiotics and anticancer drugs. Expression of OATPs, OATs and OCTs can be regulated at the protein or transcriptional level and appears to vary within each family by both protein and tissue type. All three superfamilies consist of 12 transmembrane domain proteins that have intracellular termini. Although no crystal structures have yet been determined, combinations of homology modelling and mutation experiments have been used to explore the mechanism of substrate recognition and transport. Several polymorphisms identified in members of these superfamilies have been shown to affect pharmacokinetics of their drug substrates, confirming the importance of these drug transporters for efficient pharmacological therapy. This review, unlike other reviews that focus on a single transporter family, briefly summarizes the current knowledge of all the functionally characterized human organic anion and cation drug uptake transporters of the SLCO and the SLC22A superfamilies.

The biological mechanisms underlying castration resistance of prostate cancer are not fully understood. In the present study, we examined the role of organic anion transporting polypeptides (OATPs) as importers of dehydroepiandrosterone sulfate (DHEAS) into cells to support growth under androgen-depleted conditions. Cell growth and mRNA expression of OATP genes were studied in human prostate cancer LNCaP and 22Rv1 cells under androgen-depleted conditions. The stimulatory effect of DHEAS on cell growth was investigated in LNCaP cells in which OATP1A2 had been silenced. Growth of both cell lines was stimulated by DHEAS and the effect was attenuated by STX64, an inhibitor of steroid sulfatase which can covert DHEAS to DHEA. OATP1A2 mRNA expression was increased most prominently among various genes tested in LNCaP cells grown in androgen-depleted medium. Similar results were obtained with 22Rv1 cells. Furthermore, the characteristics of [(3)H]DHEAS uptake by LNCaP cells were consistent with those of OATP-mediated transport. Knockdown of OATP1A2 in LNCaP cells resulted in loss of the DHEAS sensitivity of cell growth. Our results suggest that enhanced OATP1A2 expression is associated with adaptive cell growth of prostate cancer cells under androgen-depleted conditions. Thus, OATP1A2 may be a pharmacological target for prostate cancer treatment.

Organic anion-transporting polypeptide 1A2 (OATP1A2) (gene symbol, SLCO1A2) mediates cellular uptake of a wide range of endogenous substrates, as well as drugs and xenobiotics. OATP1A2 is expressed in several tissues, including apical membranes of small intestinal epithelial cells. Given its role in intestinal drug absorption, a detailed analysis of the mechanisms that regulate SLCO1A2 gene expression is potentially of great pharmacological relevance. We show here that treatment of human intestine-derived Caco-2 cells with vitamin D(3) markedly increased endogenous OATP1A2 mRNA and protein levels. Suppression of endogenous vitamin D receptor (VDR) expression with siRNAs significantly reduced this induction. Two alternative promoter regions exist in genomic databases for the SLCO1A2 gene. One putative VDR response element (VDRE) that was predicted to interact efficiently with VDR-retinoid X receptor α (RXRα) was identified in silico within SLCO1A2 promoter variant 1. This VDRE served as a strong binding site for the recombinant VDR-RXRα heterodimers in vitro and was potently activated by VDR in the presence of vitamin D(3) in heterologous promoter assays. In reporter assays using native promoter constructs, SLCO1A2 promoter variant 1 was strongly induced by VDR, and site-directed mutagenesis of a single VDRE within this region abolished this activation. Native VDR-RXRα also interacted with this element both in vitro and in living cells. We showed that expression of the SLCO1A2 gene is induced by vitamin D(3) at the transcriptional level through the VDR. Our results suggest that pharmacological administration of vitamin D(3) may allow modulation of intestinal absorption of OATP1A2 transport substrates.

The secretion of bile normally depends on the function of a number of membrane transport systems in hepatocytes and cholangiocytes. The transport of solutes from the blood to the bile is driven by transport systems in the plasma membrane of the basolateral and canalicular surfaces of the hepatocytes. In cholestatic animal models, the expression of hepatobiliary transporters changes in response to functional impairment of the efflux of bile salts and various organic anions. In recent years, several studies have led to an improved understanding of the function and regulation of hepatobiliary transport systems in patients with primary biliary cirrhosis (PBC). This review focuses on the adaptations in hepatobiliary transporters in PBC patients.

Sodium-dependent uptake of bile acids from blood is aliver-specific function which is mediated by theNa(+)-taurocholate cotransporting polypeptide(Ntcp). We report the stable expression of aNa(+)-taurocholate cotransporting green fluorescentfusion protein in the human hepatoblastoma cell lineHepG2, normally lacking Ntcp expression. Ntcp-EGFPassociated green fluorescence colocalized with Ntcpimmunofluorescence in the plasma membrane. Intransfected HepG2 cells, the fusion protein mediatedthe sodium-dependent uptake of the bile acidtaurocholate (K(m): 24.6 mumol/l) and of the anionicsteroids estrone-3-sulfate and dehydroepiandrosteronesulfate. We conclude that the Ntcp-EGFP fusion proteinfollows the sorting route of Ntcp, is functionallyidentical to Ntcp and could be used to monitor proteintrafficking in living HepG2 cells.

A review is presented of Gustav Paumgartner's five decades of research and practice in hepatology focusing on biliary physiology and disease. It begins with studies of the excretory function of the liver including hepatic uptake of indocyanine green, bilirubin, and bile acids. The implications of these studies for diagnosis and understanding of liver diseases are pointed out. From there, the path of scientific research leads to investigations of hepatobiliary bile acid transport and the major mechanisms of bile formation. The therapeutic effects of the hydrophilic bile acid, ursodeoxycholic acid, have greatly stimulated these studies. Although ursodeoxycholic acid therapy for dissolution of cholesterol gallstones and some other nonsurgical treatments of gallstones were largely superseded by surgical techniques, ursodeoxycholic acid is currently considered the mainstay of therapy of some chronic cholestatic liver diseases, such as primary biliary cirrhosis. The major mechanisms of action of ursodeoxycholic acid therapy in cholestatic liver diseases are discussed. An attempt is made to illustrate how scientific research can lead to advances in medical practice that help patients.

Most gene expression studies examining the effect of obesity and weight loss have been performed using adipose tissue. However, the liver also plays a central role in maintaining energy balance. We wanted to study the effects of a hypocaloric diet on overall hepatic gene expression and metabolic risk factors.

The study subjects were middle-aged, obese women. The diet intervention subjects (n=12) were on a hypocaloric, low-fat diet for 8 weeks with a daily energy intake of 5.0 MJ (1200 kcal), while the control subjects (n=19) maintained their weight. Liver biopsies were taken at the end of the diet period during a gallbladder operation. Hepatic gene expression was analyzed using microarrays by comparing the gene expression profiles from four subjects per group. A global decrease in gene expression was observed with 142 down-regulated genes and only one up-regulated gene in the diet intervention group. The diet resulted in a mean weight loss of 5% of body weight. Triglyceride and fasting insulin concentrations decreased significantly after the diet.

The global decrease in hepatic gene expression was unexpected but the results are interesting, since they included several genes not previously linked to weight reduction. However, since the comparison was made only after the weight reduction, other factors in addition to weight loss may also have been involved in the differences in gene expression between the groups. The decrease in triglyceride and fasting plasma insulin concentrations is in accordance with results from previous weight-loss studies.

Hepatic uptake and biliary excretion of organic anions (e.g., bile acids and bilirubin) is mediated by hepatobiliary transport systems. Defects in transporter expression and function can cause or maintain cholestasis and jaundice. Recruitment of alternative export transporters in coordination with phase I and II detoxifying pathways provides alternative pathways to counteract accumulation of potentially toxic biliary constituents in cholestasis. The genes encoding for organic anion uptake (NTCP, OATPs), canalicular export (BSEP, MRP2) and alternative basolateral export (MRP3, MRP4) in liver are regulated by a complex interacting network of hepatocyte nuclear factors (HNF1, 3, 4) and nuclear (orphan) receptors (e.g., FXR, PXR, CAR, RAR, LRH-1, SHP, GR). Bile acids, proinflammatory cytokines, hormones and drugs mediate causative and adaptive transporter changes at a transcriptional level by interacting with these nuclear factors and receptors. Unraveling the underlying regulatory mechanisms may therefore not only allow a better understanding of the molecular pathophysiology of cholestatic liver diseases but should also identify potential pharmacological strategies targeting these regulatory networks. This review is focused on general principles of transcriptional basolateral and canalicular transporter regulation in inflammation-induced cholestasis, ethinylestradiol- and pregnancy-associated cholestasis, obstructive cholestasis and liver regeneration. Moreover, the potential therapeutic role of nuclear receptor agonists for the management of liver diseases is highlighted.

The nuclear farnesoid X receptor (FXR) plays a pivotal role in maintaining bile acid homeostasis by regulating key genes involved in bile acid synthesis, metabolism and transport, including CYP7A1, UGT2B4, BSEP, MDR3, MRP2, ASBT, I-BABP, NTCP and OSTalpha-OSTbeta in humans. Altered expression or malfunction of these genes has been described in patients with cholestatic liver diseases. This review examines the rationale for the use of FXR ligand therapy in various cholestatic liver disorders and includes potential concerns.

We investigated transcriptional regulation of organic anion transporter OATP2B1 (SLCO2B1) that is expressed in multiple tissues such as liver, small intestine, and others and compared it with that of liver-specific OATPs.

The promoter activity was examined by luciferase assay. Specific bindings of transcription factors to the promoter region were examined by gel mobility shift assay using native and mutated nucleotides of the promoter region of OATP2B1.

Deletion-mutation study of the promoter region of OATP2B1 showed that the -59 region that included the Sp1 binding site had basal promoter activity, whereas promoter activities of the further upper region were different between intestine-derived Caco-2 cells and liver-derived HepG2 cells. The association of Sp1 to the promoter region was confirmed by gel shift assay and overexpression of Sp1 in cultured cells. Although the promoter of OATP2B1 has a putative HNF1alpha binding site, overexpression of HNF1alpha did not induce the expression of OATP2B1.

Sp1, a transcription factor, was required for constitutive expression of OATP2B1 in liver and small intestine, whereas HNF1alpha, which is involved in the expression of liver-specific OATPs, did not seem to play a role in OATP2B1 expression. Accordingly, it was suggested that the tissue expression profile of OATP2B1 was different from that of other liver-specific OATPs.

Hepatobiliary transport systems are responsible for hepatic uptake and excretion of bile salts and other biliary constituents (eg, bilirubin) into bile. Hereditary transport defects can result in progressive familial and benign recurrent intrahepatic cholestasis. Exposure to acquired cholestatic injury (eg, drugs, hormones, proinflammatory cytokines, biliary obstruction or destruction) also results in altered expression and function of hepatic uptake and excretory systems, changes that may maintain and contribute to cholestasis and jaundice. Recruitment of alternative efflux pumps and induction of phase I and II detoxifying enzymes may limit hepatic accumulation of potentially toxic biliary constituents in cholestasis by providing alternative metabolic and escape routes. These molecular changes are mediated by bile salts, proinflammatory cytokines, drugs, and hormones at a transcriptional and posttranscriptional level. Alterations of hepatobiliary transporters and enzymes are not only relevant for a better understanding of the pathophysiology of cholestatic liver diseases, but may also represent important targets for pharmacotherapy. Drugs (eg, ursodeoxycholic acid, rifampicin) used to treat cholestatic liver diseases and pruritus may counteract cholestasis via stimulation of defective transporter expression and function. In addition, therapeutic strategies may be aimed at supporting and stimulating alternative detoxification pathways and elimination routes for bile salts in cholestasis.

Recent insights into the cellular and molecular mechanisms that control the function and regulation of hepatobiliary transport have led to a greater understanding of the physiological significance of bile secretion. Individual carriers for bile acids and other organic anions in both liver and intestine have now been cloned from several species. In addition, complex networks of signals that regulate key enzymes and membrane transporters located in cells that participate in the metabolism or transport of biliary constituents are being unraveled. This knowledge has major implications for the pathogenesis of cholestatic liver diseases. Here, we review recent information on molecular aspects of hepatobiliary secretory function and its regulation in cholestasis. Potential implications of this knowledge for the design of new therapies of cholestatic disorders are also discussed.

The vectorial transport of bile salts from blood into bile is essential for the generation of bile flow, solubilization of cholesterol in bile, and emulsification of lipids in the intestine. Major transport proteins involved in the enterohepatic circulation of bile salts include the hepatocellular bile salt export pump (BSEP, ABCB11), the apical sodium-dependent bile salt transporter (ASBT, SLC10A2) in cholangiocytes and enterocytes, the sodium-dependent hepatocyte bile salt uptake system NTCP (SLC10A1), the organic anion transporting polypeptides OATP-C (SLC21A6), OATP8 (SLC21A8) and OATP-A (SLC21A3), and the multidrug resistance protein MRP3 (ABCC3). Synthesis and transport of bile salts are intricately linked processes that undergo extensive feedback and feed-forward regulation by transcriptional and posttranscriptional mechanisms. A key regulator of hepatocellular bile salt homeostasis is the bile acid receptor/farnesoid X receptor FXR, which activates transcription of the BSEP and OATP8 genes and of the small heterodimer partner 1 (SHP). SHP is a transcriptional repressor that mediates bile acid-induced repression of the bile salt uptake systems rat Ntcp and human OATP-C. A nuclear receptor that activates rodent Oatp2 (Slc21a5) and human MRP2 (ABCC2) is the pregnane X receptor/steroid X receptor PXR/SXR. Intracellular trafficking and membrane insertion of bile salt transporters is regulated by lipid, protein, and extracellular signal-related kinases in response to physiologic stimuli such as cyclic adenosine monophosphate or taurocholate. Finally, dysfunction of individual bile salt transporters such as BSEP, on account of genetic mutations, steric inhibition, suppression of gene expression, or disturbed signaling, is an important cause of cholestatic liver disease.

Molecular medicine has led to rapid advances in the characterization of hepatobiliary transport systems that determine the uptake and excretion of bile salts and other biliary constituents in the liver and extrahepatic tissues. The bile salt pool undergoes an enterohepatic circulation that is regulated by distinct bile salt transport proteins, including the canalicular bile salt export pump BSEP (ABCB11), the ileal Na(+)-dependent bile salt transporter ISBT (SLC10A2), and the hepatic sinusoidal Na(+)- taurocholate cotransporting polypeptide NTCP (SLC10A1). Other bile salt transporters include the organic anion transporting polypeptides OATPs (SLC21A) and the multidrug resistance-associated proteins 2 and 3 MRP2,3 (ABCC2,3). Bile salt transporters are also present in cholangiocytes, the renal proximal tubule, and the placenta. Expression of these transport proteins is regulated by both transcriptional and posttranscriptional events, with the former involving nuclear hormone receptors where bile salts function as specific ligands. During bile secretory failure (cholestasis), bile salt transport proteins undergo adaptive responses that serve to protect the liver from bile salt retention and which facilitate extrahepatic routes of bile salt excretion. This review is a comprehensive summary of current knowledge of the molecular characterization, function, and regulation of bile salt transporters in normal physiology and in cholestatic liver disease and liver regeneration.

Liver uptake of thyroxine (T4) is mediated by transporters and is rate limiting for hepatic 3,3',5-triiodothyronine (T3) production. We investigated whether hepatic mRNA for T4 transporters is regulated by thyroid state using Xenopus laevis oocytes as an expression system. Because X. laevis oocytes show high endogenous uptake of T4, T4 sulfamate (T4NS) was used as an alternative ligand for the hepatic T4 transporters. Oocytes were injected with 23 ng liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats, and after 3-4 days uptake was determined by incubation of injected and uninjected oocytes for 1 h at 25 degrees C or for 4 h at 18 degrees C with 10 nM [125I]T4NS. Expression of type I deiodinase (D1), which is regulated by thyroid state, was studied in the oocytes as an internal control. Uptake of T4NS showed similar approximately fourfold increases after injection of liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats. A similar lack of effect of thyroid state was observed using reverse T3 as ligand. In contrast, D1 activity induced by liver mRNA from hyperthyroid and hypothyroid rats in the oocytes was 2.4-fold higher and 2.7-fold lower, respectively, compared with euthyroid rats. Studies have shown that uptake of iodothyronines in rat liver is mediated in part by several organic anion transporters, such as the Na+/taurocholate-cotransporting polypeptide (rNTCP) and the Na-independent organic anion-transporting polypeptide (rOATP1). Therefore, the effects of thyroid state on rNTCP, rOATP1, and D1 mRNA levels in rat liver were also determined. Northern analysis showed no differences in rNTCP or rOATP1 mRNA levels between hyperthyroid and hypothyroid rats, whereas D1 mRNA levels varied widely as expected. These results suggest little effect of thyroid state on the levels of mRNA coding for T4 transporters in rat liver, including rNTCP and rOATP1. However, they do not exclude regulation of hepatic T4 transporters by thyroid hormone at the translational and posttranslational level.

The organic anion-transporting polypeptides (OATP) represent a family of proteins responsible for the membrane transport of a large number of endogenous and xenobiotic compounds with diverse chemical characteristics. OATPs are expressed in liver, kidney, brain and intestine suggesting that they may play a critical role in drug disposition. Naturally occurring polymorphisms in OATPs are currently being identified and for some, in vitro transport activities have been characterized. In this article, we review the molecular, biochemical and pharmacological aspects of known human OATPs including the presence and functional relevance of genetic polymorphisms.

Two DTPA derivatives, a mono-amide derivative containing an iodinated synthon, DTPA-IOPsp (L(1)) and the ligand DTPA(BOM)(3) (BOM=benzyloxymethyl) (L(2)), radiolabelled with (153)Sm(3+) and (111)In(3+), were studied as potential hepatospecific gamma scintigraphic agents. In vivo studies with Wistar rats show that the main excretory pathway for all the chelates studied is the hepatobiliary system. The complexes of L(2) show even greater hepatobiliary specificity than L(1), perhaps as a consequence of longer blood circulation times due to their strong affinity towards HSA. The (153)Sm(3+) chelates are also more hepatospecific than the corresponding (111)In(3+) chelates. The La(3+) and In(3+) chelates of L(1) and L(2) show some structural and dynamic differences in aqueous solution, as studied by (1)H NMR spectroscopy. While only two nona-coordinated isomers were observed for the La(3+) complexes with both ligands, its number is much larger in the In(3+) complexes, with both octa- and hepta-coordinated species (with unbound side arms), as well as structural isomers for each coordination number.

Primary sclerosing cholangitis (PSC) is a cholestatic liver disease of unknown etiology. Although the primary defect affects cholangiocytes, cholestatic injury of hepatocytes may promote further liver damage. Since down-regulation of hepatocellular organic anion transporters is implicated in the molecular pathogenesis of cholestasis, expression of these transporters was determined in a novel rat model, which closely resembles human PSC.

Hepatic protein and mRNA expression of basolateral (Ntcp, Oatp1, 2 and 4) and canalicular (Mrp2, Bsep) organic anion transporters were analyzed 1, 4 and 12 weeks after induction of experimental PSC by 2,4,6-trinitrobenzenesulfonic acid (TNBS).

Specific down-regulation of basolateral and canalicular transport systems except Oatp4 and Bsep proteins occurred during the acute phase of inflammation. In chronic cholangitis 12 weeks after TNBS Mrp2 protein and mRNA remained down-regulated by 40-50% of controls (P<0.05). In addition Oatp1 protein was also reduced by 40+/-13% (P<0.05), whereas all other transporters returned to control values.

In chronic cholangitis only canalicular Mrp2 expression remained down-regulated. This might represent the first injury to hepatocytes in chronic cholangitis as an extension of liver injury from the level of cholangiocytes to hepatocytes in PSC.

Bile salts are the major organic solutes in bile and undergo extensive enterohepatic circulation. Hepatocellular bile salt uptake is mediated predominantly by the Na(+)-taurocholate cotransport proteins Ntcp (rodents) and NTCP (humans) and by the Na(+)-independent organic anion-transporting polypeptides Oatp1, Oatp2, and Oatp4 (rodents) and OATP-C (humans). After diffusion (bound by intracellular bile salt-binding proteins) to the canalicular membrane, monoanionic bile salts are secreted into bile canaliculi by the bile salt export pump Bsep (rodents) or BSEP (humans). Both belong to the ATP-binding cassette (ABC) transporter superfamily. Dianionic conjugated bile salts are secreted into bile by the multidrug-resistance-associated proteins Mrp2/MRP2. In bile ductules, a minor portion of protonated bile acids and monomeric bile salts are reabsorbed by non-ionic diffusion and the apical sodium-dependent bile salt transporter Asbt/ASBT, transported back into the periductular capillary plexus by Mrp3/MRP3 [and/or a truncated form of Asbt (tAsbt)], and subjected to cholehepatic shunting. The major portion of biliary bile salts is aggregated into mixed micelles and transported into the intestine, where they are reabsorbed by apical Oatp3, the apical sodium-dependent bile salt transporter (ASBT), cytosolic intestinal bile acid-binding protein (IBABP), and basolateral Mrp3/MRP3 and tAsbt. Transcriptional and posttranscriptional regulation of these enterohepatic bile salt transporters is closely related to the regulation of lipid and cholesterol homeostasis. Furthermore, defective expression and function of bile salt transporters have been recognized as important causes for various cholestatic liver diseases.

The biological half-life (t1/2) of perfluorooctanoic acid (PFOA) in male rats is 70 times longer than that in female rats. The difference is mainly due to the difference in renal clearance (CL(R)), which was significantly reduced by probenecid, suggesting that PFOA is excreted by organic anion transporter(s). Castration of male rats caused a 14-fold increase in the CL(R) of PFOA, which made it comparable with that of female rats. The elevated PFOA CL(R) in castrated males was reduced by treating them with testosterone. Treatment of male rats with estradiol increased the CL(R) of PFOA. In female rats, ovariectomy caused a significant increase in CL(R) of PFOA, which was reduced by estradiol treatment. Treatments of female rats with testosterone reduced the CL(R) of PFOA as observed in castrated male rats. To identify the transporter molecules that are responsible for PFOA transport in rat kidney, renal mRNA levels of organic anion transporter 1 (OAT1), OAT2, OAT3, organic anion transporting polypeptide 1 (oatp1), oatp2 and kidney specific organic anion transporter (OAT-K) were determined in male and female rats under various hormonal states and compared with the CL(R) of PFOA. The level of OAT2 mRNA in male rats was only 13% that in female rats. Castration or estradiol treatment increased the level of OAT2 mRNA whereas treatment of castrated male rats with testosterone reduced it. In contrast to OAT2, mRNA levels of both oatp1 and OAT-K were significantly higher in male rats compared with female rats. Castration or estradiol treatment caused a reduction in the levels of mRNA of oatp1 and OAT-K in male rats. Ovariectomy of female rats significantly increased the level of OAT3 mRNA. Multiple regression analysis suggests that the change in the CL(R) of PFOA is, at least in part, due to altered expression of OAT2 and OAT3.

OATP-C (SLC21A6) is the predominant Na(+)-independent uptake system for bile salts and bilirubin of human liver and is expressed exclusively at the basolateral (sinusoidal) hepatocyte membrane. To investigate the basis of liver-specific expression of OATP-C, we studied promoter function in the two hepatocyte-derived cell lines HepG2 and Huh7 and in nonhepatic HeLa cells. OATP-C promoter constructs containing from 66 to 950 nucleotides of 5'-regulatory sequence were active in HepG2 and Huh7 but not HeLa cells, indicating that determinants of hepatocyte-specific expression reside within the minimal promoter. Deoxyribonuclease I footprint analysis revealed a single region that was protected by HepG2 and Huh7 but not HeLa cell nuclear extracts. The liver-enriched transcription factor hepatocyte nuclear factor 1 alpha (HNF1 alpha) was shown by mobility shift assays to bind within this footprint. Coexpression of HNF1 alpha stimulated OATP-C promoter activity 30-fold in HepG2 and 49-fold in HeLa cells. Mutation of the HNF1 site abolished promoter function, indicating that HNF1 alpha is critical for hepatocyte-specific OATP-C gene expression. The human OATP8 (SLC21A8) and mouse Oatp4 (Slc21a6) promoters were also responsive to HNF1 alpha coexpression in HepG2 cells. These data support a role for HNF1 alpha as a global regulator of liver-specific bile salt and organic anion transporter genes.

In chronic cholestatic liver diseases, biliary excretion of organic anions from blood into bile is impaired. The aim of this study was to identify the underlying mechanism.

Expression of the basolateral organic anion transporting polypeptide OATP-C (SLC21A6) and the canalicular multidrug resistance protein 2 (MRP2) was studied in patients with primary sclerosing cholangitis (PSC) (n=4), a chronic cholestatic liver disease, and in non-cholestatic controls (n=4) (two with chronic hepatitis C, one with idiopathic liver cirrhosis and one with fatty liver). Total RNA was isolated from liver tissue, reverse transcribed and subjected to polymerase chain reaction (PCR) amplification using primers specific for OATP-C, MRP2 and beta-actin. PCR products were quantified densitometrically.

When normalized for beta-actin expression, the level of OATP-C mRNA in liver tissue of patients with PSC was 49% of controls (OATP-C/beta-actin 1.60+/-0.25 vs. 3.24+/-0.69; p<0.05) and the level of MRP2 mRNA was 27% of controls (MRP2/beta-actin 0.70+/-0.36 vs. 2.54+/-0.56; p<0.01).

Both OATP-C and MRP2 are decreased as measured by mRNA level in PSC. Downregulation of OATP-C might be the consequence of impaired canalicular secretion of organic anions and could serve to reduce the organic anion load of cholestatic hepatocytes.

To determine the contribution of the C282Y and H63D mutations in the HFE gene to clinical expression of hereditary hemochromatosis.

Pooled analysis of 14 case-control studies reporting HFE genotype data, to evaluate the association of different HFE genotypes with iron overload. In addition, we used data from the pooled analysis and published data to estimate the penetrance of the C282Y/C282Y genotype.

Homozygosity for the C282Y mutation carried the largest risk for iron overload (OR = 4383, 95% CI 1374 to >10,000) and accounted for the majority of hemochromatosis cases (attributable fraction (AF) = 0.73). Risks for other genotypes were much smaller: OR = 32 for genotype C282Y/H63D (95% CI 18.5 to 55.4, AF = 0.06); OR = 5.7 for H63D/H63D (95% CI 3.2 to 10.1, AF = 0.01); OR = 4.1 for C282Y heterozygosity (95% CI 2.9 to 5.8, with heterogeneity in study results, making this association uncertain); and OR = 1.6 for H63D heterozygosity (95% CI 1 to 2.6, AF = 0.03). Estimates of penetrance for the C282Y/C282Y genotype were highly sensitive to estimates of the prevalence of iron overload disease. At a prevalence of 2.5 per 1000 or less, penetrance of the C282Y/C282Y genotype is unlikely to exceed 50%. Penetrance of other HFE genotypes is much lower.

C282Y homozygosity confers the highest risk for iron overload but the H63D mutation is also associated with increased risk. Our data indicate a gradient of risk associated with different HFE genotypes and thus suggest the presence of other modifiers, either genetic or environmental, that contribute to the clinical expression of hemochromatosis.

Cholestasis in primary biliary cirrhosis results from impairment of bile flow either by reduced transport at the level of the canaliculi or by disturbed bile flow through damaged intrahepatic bile ductules. Whatever its cause, the expression of hepatic transport proteins will be affected. In cholestatic rats: the expression of the multispecific organic anion transporter mrp2 is decreased; the bile salt export pump bsep and the phospholipid transporter mdr2 are less affected; the carrier protein for hepatic uptake of bile salts ntcp is sharply down-regulated; Mrp3, a basolateral ATP-dependent transporter for glucuronides and bile salts, is upregulated. Thus, bile salts that cannot exit the hepatocyte because of the cholestasis are effectively removed across the basolateral membrane. These may be adaptive responses in defence against overloading of hepatocytes with cytotoxic bile salts. These responses show that the expression of hepatic transporter proteins is highly regulated. This occurs by transcriptional and post-transcriptional mechanisms. Primary biliary cirrhosis starts as a disease of the small intrahepatic bile ducts and therefore the experimental evidence for 'cross-talk' between hepatocytes and cholangiocytes is of great interest for this disease and needs to be further investigated. New insights in bile physiology may enable the development of new therapies for cholestatic liver diseases as primary biliary cirrhosis.

Based on sequence homology to the human organic anion transporting polypeptide 2 (OATP2; SLC21A6), we cloned a new member of the SLC21A superfamily of solute carriers, termed OATP8 (SLC21A8). The protein of 702 amino acids showed an amino acid identity of 80% with human OATP2. Based on Northern blotting, the expression of OATP8 was restricted to human liver. Cosmid clones containing the genes encoding human OATP1 (SLC21A3), OATP2 (SLC21A6), and OATP8 (SLC21A8) served to establish their genomic organization. All three genes contained 14 exons with 13 identical splice sites when transferred to the amino acid sequence. An antibody raised against the carboxyl terminus localized OATP8 to the basolateral membrane of human hepatocytes and the recombinant glycoprotein, expressed in MDCKII cells, to the lateral membrane. Transport properties of OATP8 were studied in stably transfected MDCKII and HEK293 cells. Organic anions transported by human OATP8 included sulfobromophthalein, with a K(m) of 3.3 microm, and 17beta-glucuronosyl estradiol, with a K(m) of 5.4 microm. Several bile salts were not substrates. Thus, human OATP8 is a new uptake transporter in the basolateral hepatocyte membrane with an overlapping but distinct substrate specificity as compared with OATP2, which is localized to the same membrane domain.

We have cloned a cDNA that codes for mouse liver-specific transporter-1, mouse lst-1. The cDNA is comprised of 3296 base pairs and it contains a coding sequence for a protein of 689 amino acids with 12 putative transmembrane domains. The deduced amino acid sequence of the mouse lst-1 shares 64 and 77% identities with the reported human and rat lsts, respectively. Northern blot analysis demonstrates that mouse lst-1 mRNA is expressed exclusively in liver. We also report here the structural organization of the mouse lst-1 gene as the first evidence for the structure of a gene encoding an lst. The mouse lst-1 gene spans approximately 60 kbp in length and consists of 16 exons, including two noncoding exons. All the introns are flanked by GT-AG consensus splice sequences. 5'-Rapid Amplification of cDNA Ends (RACE) analyses demonstrate three splice variant mRNAs involving the noncoding exon 2 and exon 3. The 5'-flanking region of the gene contains consensus CAAT and TATA boxes and several potential binding sites for transcription factors for CAAT enhancer binding protein (C/EBP) and hepatocyte nuclear factors (HNF-3beta, HFH-1, and HFH-2), transcription factors important for liver-specific gene expression.

From the multiple mechanisms of cholestasis presented in this article, a unifying hypothesis may be deduced by parsimony. The disturbance of the flow of bile must inevitably lead to the intracellular retention of biliary constituents. Alternatively, the lack of specific components of bile unmasks the toxic potential of other components, as in the case of experimental mdr2 deficiency. In the sequence of events that leads to liver injury, the cytotoxic action of bile salts is pivotal to all forms of cholestasis. The inhibition of the bsep by drugs, sex steroids, or monohydroxy bile salts is an example of direct toxicity to the key mediator in canalicular bile salt excretion. In other syndromes, the dysfunction of distinct hepatocellular transport systems is the primary pathogenetic defect leading to cholestasis. Such dysfunctions include the genetic defects in PFIC and the direct inhibition of gene transcription by cytokines. Perturbations in the short-term regulation of transport protein function are exemplified by the cholestasis of endotoxinemia. The effect of bile salts on signal transduction, gene transcription, and transport processes in hepatocytes and cholangiocytes has become the focus of intense research in recent years. The central role of bile salts in the pathogenesis of cholestasis has, ironically, become all the more evident from the improvement of many cholestatic syndromes with oral bile salt therapy.

The alterations of hepatobiliary transport that occur in cholestasis can be divided into primary defects, such as mutations of transporter genes or acquired dysfunctions of transport systems that cause defective canalicular or cholangiocellular secretion, and secondary defects, which result from biliary obstruction. The dysfunction of distinct biliary transport systems as a primary cause of cholestasis is exemplified by the genetic defects in progressive familial intrahepatic cholestasis or by the direct inhibition of transporter gene expression by cytokines. In both, the hepatocellular accumulation of toxic cholephilic compounds causes multiple alterations of hepatocellular transporter expression. In addition, lack of specific components of bile caused by a defective transporter, as in the case of mdr2/MDR3 deficiency, unmasks the toxic potential of other components. The production of bile is critically dependent upon the coordinated regulation and function of sinusoidal and canalicular transporters, for instance of Na+-taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP). Whereas the downregulation of the unidirectional sinusoidal uptake system NTCP protects the hepatocyte from further intracellular accumulation of bile salts, the relative preservation of canalicular BSEP expression serves to uphold bile salt secretion, even in complete biliary obstruction. Conversely, the strong downregulation of canalicular MRP2 (MRP, multidrug resistance protein) in cholestasis forces the hepatocyte to upregulate basolateral efflux systems such as MRP3 and MRP1, indicating an inverse regulation of basolateral and apical transporters The regulation of hepatocellular transporters in cholestasis adheres to the law of parsimony, since many of the cellular mechanisms are pivotally governed by the effect of bile salts. The discovery that bile salts are the natural ligand of the farnesoid X receptor has shown us how the major bile component is able to regulate its own enterohepatic circulation by affecting transcription of the genes critically involved in transport and metabolism.

We found previously that expression of multidrug resistance-associated protein (MRP) 3 is induced in a mutant rat strain (Eisai hyperbilirubinemic rats) whose canalicular multispecific organic anion transporter (cMOAT/MRP2) function is hereditarily defective and in normal Sprague-Dawley (SD) rats after ligation of the common bile duct. In the present study, the inducible nature of MRP3 was examined, using Northern and Western blot analyses, in comparison with that of other secondary active [Na(+)-taurocholic acid cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (oatp1), and organic cation transporter (OCT1)] and primary active [P-glycoprotein (P-gp), cMOAT/MRP2, and MRP6] transporters. alpha-Naphthylisothiocyanate treatment and common bile duct ligation induced expression of P-gp and MRP3, whereas expression of Ntcp, oatp1, and OCT1 was reduced by the same treatment. Although expression of MRP3 was also induced by administration of phenobarbital, that of cMOAT/MRP2, MRP1, and MRP6 was not affected by any of these treatments. Moreover, the mRNA level of MRP3, but not that of P-gp, was increased in SD rats after administration of bilirubin and in Gunn rats whose hepatic bilirubin concentration is elevated because of a defect in the expression of UDP-glucuronosyl transferase. However, the MRP3 protein level was not affected by bilirubin administration. Although the increased MRP3 mRNA level was associated with the increased concentration of bilirubin and/or its glucuronides in mutant rats and in SD rats that had undergone common bile duct ligation or alpha-naphthylisothiocyanate treatment, we must assume that factor(s) other than these physiological substances are also involved in the increased protein level of MRP3.

This study assessed the expression of the recently identified adenosine triphosphate-dependent bile salt export pump and the functional ability to excrete bile salts in cholestatic models in the rat.

The effects of common bile duct ligation, endotoxin, and ethinylestradiol on bile salt export pump messenger RNA levels, protein expression, and tissue localization were determined. Changes in the expression of 3 other hepatocyte membrane transporters (Na(+) taurocholate cotransporter, multispecific organic anion transporter, and P-glycoprotein) were also determined for comparison. Functional assessment of bile salt excretion was determined after bile duct ligation.

Expression of the bile salt export pump was diminished but relatively preserved compared with other membrane transporters. Tissue localization of the bile salt export pump persisted at the canalicular domain in all 3 models. In contrast, expressions of the Na(+) taurocholate cotransporter and multispecific organic anion transporter were more profoundly diminished. P-glycoprotein levels increased severalfold with common bile duct ligation but were unchanged with either endotoxin or ethinylestradiol. The capacity to excrete bile salts was relatively maintained 3 and even 14 days after bile duct ligation.

Alterations in expression of the bile salt export pump may account for the functional alterations of bile salt secretion observed in cholestasis. However, relative preservation of expression is associated with persistent bile salt excretion and may lessen the extent of liver injury produced by bile salt retention.

The mechanisms were investigated for the hepatic transport of 4 different gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI). In basolateral rat hepatocyte plasma membrane vesicles, Gd-DTPA uptake was indistinguishable from non-specific binding to vesicles; Gd-BOPTA and Gd-EOB-DTPA entered plasma membrane vesicles following a linear, concentration-dependent mechanism up to 1.5 mM of substrate. By contrast, Gd-B 20790 uptake followed a saturative kinetic with an apparent Km of 92 +/- 15 microM and a Vmax of 143 +/- 42 pmol/mg prot/15 sec, and it occurred into an osmotic-sensitive space. Sulfobromophthalein ant taurocholate, but not unconjugated bilirubin inhibited the uptake rate of Gd-B 20790 but not that of the other three compounds. Injection into Xenopus laevis oocytes of 5 ng of human OATP cRNA resulted, after 3 days, in a >/=2-fold stimulation (p < 0.001) of transport of Gd-B 20790 but not of Gd-BOPTA or Gd-EOB-DTPA. Collectively, these data indicate that the hepatic uptake of the MRI contrast agent Gd-B 20790 is a carrier-mediated mechanism operated by OATP while MRI compounds with other chemical structures enter the hepatocyte by other mechanisms.