Drug-induced cholestasis (DIC) is a leading cause of drug-induced liver injury post-drug marketing, characterized by bile flow obstruction and toxic bile constituent accumulation within hepatocytes. This study investigates the toxicity associated with intracellular bile acid (BA) accumulation during DIC development. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, we examined intracellular BA concentrations in human precision-cut liver slices (PCLS) following the administration of cyclosporin A and chlorpromazine, both with and without an established BA mixture. Our findings indicate toxicity of cyclosporin A upon BA addition, while chlorpromazine's toxicity remained unaffected. Although neither drug led to the accumulation of all BAs intracellularly, BA mixture addition resulted in the accumulation of unconjugated BAs associated with DIC, such as deoxycholic acid (DCA) and cholic acid (CA). Additionally, cyclosporin A increased taurolithocholic acid (TLCA) concentrations. In the absence of the BA mixture, a decrease in conjugated BAs was observed, suggesting inhibition of BA metabolism by cholestatic drugs and warranting further investigation. The evident increase in CA and DCA for both drugs (and TLCA for cyclosporin A), despite not exacerbating toxicity with chlorpromazine, suggests these increases may be related to DIC development and possible toxicity. In conclusion, the current human PCLS model is appropriate for investigating and detecting essential contributors to DIC and can be used in future studies elucidating DIC ex vivo.

Cholestatic liver injury can lead to serious symptoms and prognoses in the clinic. Currently, an effective medical treatment is not available for cholestatic liver injury. Human menstrual blood-derived stem cells (MenSCs) are considered as an emerging treatment in various diseases. This study aimed to explore the treatment effect of MenSCs in cholestatic liver injury.

The treatment effect of MenSCs on chronic cholestatic liver injury was verified in 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC)-induced C57/BL6 mice. Pathological, fibrosis area in the liver tissue and serum liver enzymes were tested. Proteomics and western blot were used to explore the related targets and molecular mechanisms. Adeno-associated virus (AAV) 9-infected mice were applied for verification.

MenSCs markedly improved the survival rate of the DDC-treated mice (60% vs. 100%), and decreased the mouse serum aspartate aminotransferase (AST) (169.4 vs. 108.0 U/L, p < 0.001), alanine aminotransferase (ALT) (279.0 vs. 228.9 U/L, p < 0.01), alkaline phosphatase (ALP) (45.6 vs. 10.6 U/L, p < 0.0001), direct bilirubin (DBIL) (108.3 vs. 14.0 μmol/L, p < 0.0001) and total bilirubin (TBIL) (179.2 vs. 43.3 μmol/L, p < 0.0001) levels as well as intrahepatic cholestasis, bile duct dilation and fibrotic areas (16.12 vs. 6.57%, p < 0.05). The results further indicated that MenSCs repaired the DDC-induced liver tight junction (TJ) pathway and bile transporter (OATP2, BSEP and NTCP1) injury, thereby inhibiting COL1A1, α-SMA and TGF-β1 activation by upregulating liver β-catenin expression.

MenSC transplantation could be an effective treatment method for cholestatic liver injury in mice. MenSCs may exhibit therapeutic effects by regulating β-catenin expression.

The tumour suppressor p53 is traditionally recognized as a surveillance molecule to preserve genome integrity. Recent studies have demonstrated that it contributes to metabolic diseases. Here, we investigated the role of p53 in the regulation of bile acid disposition and cholestasis.

The bile acid disposition-related gene expression profile affected by p53 activation was assessed in mouse primary hepatocytes with p53 depletion and in Trp53-null mice. Dual luciferase reporter assay was used to detect the transcriptional activities of target genes. Anticholestatic effects of p53 activator doxorubicin were investigated in a 0.5% cholic acid-fed mouse model of cholestasis. Changes in bile acids were evaluated using metabolomics analysis.

Doxorubicin-mediated p53 activation induced Cyp2b10, Sult2a1 and Abcc2/3/4 expression in mice in vitro and in vivo. ABCC3 and CYP2B6 (human orthologue of Cyp2b10) were identified as direct p53 target genes. Doxorubicin attenuated cholic acid-induced cholestasis in mice, as demonstrated by shrunken gall bladder, decreased serum total bile acid and total bilirubin levels and alkaline phosphatase activity. Targeted metabolomics analysis revealed that doxorubicin enhanced the excretion of bile acid metabolites from serum and liver to intestine and faeces. Up-regulation of Cyp2b10, Sult2a1 and Abcc2/3/4 expression was further confirmed in cholestatic mice. Cholic acid-induced cholestatic injury was aggravated in p53-deficient mice and levels of bile acid in intestine and faeces were decreased.

Our findings suggest a novel role of p53 in promoting bile acid disposition and alleviating cholestatic syndrome, which provides a potential therapeutic target for cholestasis.

Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme, but the factors governing transcriptional regulation of its expression remain poorly understood. Based on previous reports of small heterodimer partner (SHP) playing an important role as a transcriptional repressor of CYP2D6 expression, here we investigated how a known upstream regulator of SHP expression, namely cholestasis triggered by cholic acid (CA) feeding in mice, can lead to altered CYP2D6 expression. To this end, CYP2D6-humanized (Tg-CYP2D6) mice were fed with a CA-supplemented or control diet for 14 days, and hepatic expression of multiple genes was examined. Unexpectedly, CA feeding led to insignificant changes in SHP mRNA but also to significant (2.8-fold) decreases in SHP protein levels. In silico analysis of the SHP gene regulatory region revealed a putative binding site for a microRNA, miR-142-3p. Results from luciferase reporter assays suggest that miR-142-3p targets the SHP gene. Hepatic expression of miR-142-3p was significantly increased in CA-fed mice (∼5-fold), suggesting a potential role of miR-142-3p in the regulation of SHP expression in cholestasis. The decreased SHP protein levels were accompanied by increased expression and activity of CYP2D6 in the liver of CA-fed mice. These results suggest potential roles of differential hepatic levels of bile acids in the transcriptional regulation of CYP2D6 expression.

Alpha-naphthylisothiocyanate (ANIT) is a toxicant that is widely used in rodents to model human intrahepatic cholestasis. The aim of the study is to investigate whether effects of dioscin on ANIT-induced cholestasis are related to changes in expression of hepatic transporters in rats. Effects of dioscin on cholestasis were examined by histology and biochemical marker levels. The functional changes of hepatic transporters were determined by in vitro, in situ and in vivo. qRT-PCR and western blot were used to assess the expression of hepatic transporters in cholestatic rats. Dioscin administration could ameliorate cholestasis, as evidenced by reduced biochemical markers as well as improved liver pathology. The uptakes of organic anion transporting polypeptide (Oatp) substrates were altered in liver uptake index in vivo, perfused rat liver in situ and isolated rat hepatocytes in vitro in cholestasis rats. qRT-PCR and western blot analysis indicated co-treatment of ANIT with dioscin prevented the adaptive down-regulation of Oatp1a1, 1b2, and prompted the up-regulation of Oatp1a4, multidrug resistance-associated protein (Mrp) 2 and bile salt export pump (Bsep). In addition, concerted effects on Mrp2 and Bsep occurred through up-regulation of small heterodimer partner by activating farnesoid X receptor. Dioscin might prevent impairment of hepatic function by restoring hepatic transporter expression.

Bariatric surgery is the most effective and durable treatment option for obesity today. More importantly, beyond weight loss, bariatric procedures have many advantageous metabolic effects including reversal of obesity-related liver disease--nonalcoholic steatohepatitis (NASH). NASH is an important comorbidity of obesity given that it is a precursor to the development of liver cirrhosis that may necessitate liver transplantation in the long run. Simultaneously, we and others have observed increased serum bile acids in humans and animals that undergo bariatric surgery. Specifically, our preclinical studies have included experimental procedures such as 'ileal transposition' or bile diversion and established procedures such as Roux-en-Y gastric bypass and the adjustable gastric band. Importantly, these effects are not simply the result of weight loss since our data show that the resolution of NASH and increase in serum bile acids are not seen in rodents that lose an equivalent amount of weight via food restriction. In particular, we have studied the role of altered bile acid signaling, in the potent impact of a bariatric procedure termed 'vertical sleeve gastrectomy' (VSG). In this review we focus on the mechanisms of NASH resolution and weight loss after VSG surgery. We highlight the fact that bariatric surgeries can be used as 'laboratories' to dissect the mechanisms by which these procedures work to improve obesity and fatty liver disease. We describe key bile acid signaling elements that may provide potential therapeutic targets for 'bariatric-mimetic technologies' that could produce benefits similar to bariatric surgery--but without the surgery!

Metabolomics is comprehensive analysis of low-molecular-weight endogenous metabolites in a biological sample. It could enable mapping of perturbations of early biochemical changes in diseases and hence provide an opportunity to develop predictive biomarkers that could provide valuable insights into the mechanisms of diseases. The aim of this study was to elucidate the changes in endogenous metabolites and to phenotype the metabolic profiling of d-galactosamine (GalN)-inducing acute hepatitis in rats by UPLC-ESI MS.

The systemic biochemical actions of GalN administration (ip, 400 mg/kg) have been investigated in male wistar rats using conventional clinical chemistry, liver histopathology and metabolomic analysis of UPLC- ESI MS of urine. The urine was collected predose (-24 to 0 h) and 0-24, 24-48, 48-72, 72-96 h post-dose. Mass spectrometry of the urine was analysed visually and via conjunction with multivariate data analysis.

Results demonstrated that there was a time-dependent biochemical effect of GalN dosed on the levels of a range of low-molecular-weight metabolites in urine, which was correlated with developing phase of the GalN-inducing acute hepatitis. Urinary excretion of beta-hydroxybutanoic acid and citric acid was decreased following GalN dosing, whereas that of glycocholic acid, indole-3-acetic acid, sphinganine, n-acetyl-l-phenylalanine, cholic acid and creatinine excretion was increased, which suggests that several key metabolic pathways such as energy metabolism, lipid metabolism and amino acid metabolism were perturbed by GalN.

This metabolomic investigation demonstrates that this robust non-invasive tool offers insight into the metabolic states of diseases.

Our objective was to investigate the role of bile acids in hepatic steatosis reduction after vertical sleeve gastrectomy (VSG).

High fat diet (HFD)-induced obese C57Bl/6 mice were randomized to VSG, Sham operation (Sham), Sham operation with pair feeding to VSG (Sham-PF), or nonsurgical controls (Naïve). All mice were on HFD until sacrifice. Mice were observed postsurgery and data for body weight, body composition, metabolic parameters, serum bile acid level and composition were collected. Further hepatic gene expression by mRNA-seq and RT-PCR analysis was assessed.

VSG and Sham-PF mice lost equal weight postsurgery while VSG mice had the lowest hepatic triglyceride content at sacrifice. The VSG mice had elevated serum bile acid levels that positively correlated with maximal weight loss. Serum bile composition in the VSG group had increased cholic and tauroursodeoxycholic acid. These bile acid composition changes in VSG mice explained observed downregulation of hepatic lipogenic and bile acid synthetic genes.

VSG in obese mice results in greater hepatic steatosis reduction than seen with caloric restriction alone. VSG surgery increases serum bile acids that correlate with weight lost postsurgery and changes serum bile composition that could explain suppression of hepatic genes responsible for lipogenesis.

Green tea has been purported to have beneficial health effects including protective effects against oxidative stress. Acetaminophen (APAP) is a widely used analgesic drug that can cause acute liver injury in overdose situations. These studies explored the effects of green tea extract (GTE) on APAP-induced hepatotoxicity in liver tissue extracts using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. Mice were orally administered GTE, APAP or GTE and APAP under three scenarios. APAP alone caused a high degree of hepatocyte necrosis associated with increases in serum transaminases and alterations in multiple metabolic pathways. The time of GTE oral administration relative to APAP either protected against or potentiated the APAP-induced hepatotoxicity. Dose dependent decreases in histopathology scores and serum transaminases were noted when GTE was administered prior to APAP; whereas, the opposite occurred when GTE was administered after APAP. Similarly, metabolites altered by APAP alone were less changed when GTE was given prior to APAP. Significantly altered pathways included fatty acid metabolism, glycerophospholipid metabolism, glutathione metabolism, and energy pathways. These studies demonstrate the complex interaction between GTE and APAP and the need to employ novel analytical strategies to understand the effects of dietary supplements on pharmaceutical compounds.

The administration of a sodium cholate-supplemented high-fat (CAHF) diet in mice induced the predominant accumulation of cholesterol esters (CE) in the liver and biochemical and histological features of liver injury. Cholesteryl oleate was the most abundant CE found in the liver of the mice fed the CAHF diet. We examined the effect of ethyl eicosapentaenoate (EPA) on hepatic CE accumulation and liver injury in the mice fed the CAHF diet. The EPA supplementation suppressed the elevation in the level of cholesteryl oleate in the liver. The expression levels of sterol O-acyltransferase-2 and stearoyl-CoA desaturase-1 mRNA in the liver were elevated in the mice fed the CAHF diet, but they were normalized by the EPA supplementation. However, the elevation in serum transaminase activity, the sign of inflammatory cell exudation and inflammatory gene responses in the liver of the mice fed the EPA-supplemented diet were enhanced compared with those of the mice fed the CAHF diet. We demonstrated that EPA supplementation attenuated CE accumulation but aggravated liver injury and liver inflammation in the mice fed the CAHF diet.

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

Liver toxicity represents an important healthcare issue because it causes significant morbidity and mortality and can be difficult to predict before symptoms appear owing to drug therapy or exposure to toxicants. Using metabolomic techniques, we discovered common biomarkers for the prediction of hepatotoxicity in rat urine using mass spectrometry. For this purpose, liver toxicity was induced by 5 days of oral administration of carbon tetrachloride (1 ml kg(-1) per day), acetaminophen (1000 mg kg(-1) per day) and methotrexate (50 mg kg(-1) per day). Serum levels of alkaline phosphatase aspartate aminotransferase, alanine aminotransferase and histopathology in liver tissue were then checked to demonstrate liver toxicity. Global metabolic profiling with UPLC-TOF-MS (ultraperformance liquid chromatography-mass spectrometry), multivariate analysis (partial least square-discriminant analysis, hierarchical analysis) and database searching were performed to discover common biomarkers for liver toxicity induced by these three compounds. Urinary concentrations of the newly discovered biomarkers were then quantified to confirm them as biomarkers of hepatotoxicity with targeted metabolic profiling using GC (gas chromatography)-MS and CE (capillary electrophoresis)-MS. In the results, steroids, amino acids and bile acids were metabolically changed between the control and drug-treated groups in global metabolic profiling; 11β-hydroxyandrosterone, epiandrosterone, estrone, 11-dehydrocorticosterone, glycine, alanine, valine, leucine, dl-ornithine, 3-methylhistidine, cholic acid and lithocholic acid were selected as liver toxicity biomarkers after performing targeted metabolic profiling. In conclusion, we discovered metabolite biomarkers belonging to three different metabolic pathways to check for liver toxicity with mass spectrometry from a metabolomics study that could be used to evaluate hepatotoxicity induced by drugs or other toxic compounds.

The farnesoid X receptor/retinoid X receptor-alpha (FXR/RXRalpha) complex regulates bile salt homeostasis, in part by modulating transcription of the bile salt export pump (BSEP/ABCB11) and small heterodimer partner (SHP/NR0B2). FXR is activated by bile salts, RXRalpha by the vitamin A derivative 9-cis retinoic acid (9cRA). Cholestasis is associated with vitamin A malabsorption. Therefore, we evaluated the role of vitamin A/9cRA in the expression of human and mouse bile salt export pump (hBSEP/mBsep), small heterodimer partner (hSHP/mShp), and mouse sodium-dependent taurocholate co-transporting polypeptide (mNtcp). HBSEP and hSHP transcription were analyzed in FXR/RXRalpha-transfected HepG2 cells exposed to chenodeoxycholic acid (CDCA) and/or 9cRA. BSEP promoter activity was determined by luciferase reporter assays, DNA-binding of FXR and RXRalpha by pull-down assays. Serum bile salt levels and hepatic expression of Bsep, Shp, and Ntcp were determined in vitamin A-deficient (VAD)/cholic acid (CA)-fed C57BL/6J mice. Results indicated that 9cRA strongly repressed the CDCA-induced BSEP transcription in HepG2 cells, whereas it super-induced SHP transcription; 9cRA reduced DNA-binding of FXR and RXRalpha. The 9cRA repressed the CDCA-induced BSEP promoter activity irrespective of the exact sequence of the FXR-binding site. In vivo, highest Bsep messenger RNA (mRNA), and protein expression was observed in CA-fed VAD mice. Shp transcription was highest in CA-fed vitamin A-sufficient mice. Ntcp protein expression was strongly reduced in CA-fed VAD mice, whereas mRNA levels were normal. CA-fed control and VAD mice had similarly increased serum bile salt levels.

We showed that 9cRA has opposite effects on bile salt-activated transcription of FXR/RXRalpha target genes. Vitamin A deficiency in CA-fed mice leads to high BSEP expression. Clearance of serum bile salts may, however, be limited because of post-transcriptional reduction of Ntcp. The molecular effects of vitamin A supplementation during cholestasis need further analysis to predict a therapeutic effect.

The disposition of most drugs is highly dependent on specialized transporters. OAT1 and OAT3 are two organic anion transporters expressed in the basolateral membrane of renal proximal tubule cells, identified as contributors to xenobiotic and endogenous organic anion secretion. It is well known that cholestasis may cause renal damage. Impairment of kidney function produces modifications in the renal elimination of drugs. Recent studies have demonstrated that the renal abundance of OAT1 and OAT3 plays an important role in the renal elimination of organic anions in the presence of extrahepatic cholestasis. Time elapsed after obstructive cholestasis has an important impact on the regulation of both types of organic anion transporters. The renal expression of OAT1 and OAT3 should be taken into account in order to improve pharmacotherapeutic efficacy and to prevent drug toxicity during the onset of this hepatic disease.

Lithocholic acid is a lipid-soluble hepatotoxic bile acid that accumulates in the liver during cholestasis. A potential detoxification pathway for lithocholic acid involves hydroxylation by hepatic cytochrome P450 (P450) enzymes. The purpose of the present study was to identify the hepatic microsomal metabolites of lithocholic acid by liquid chromatography/mass spectrometry and to determine the P450 enzymes involved. Incubation of lithocholic acid with rat hepatic microsomes and NADPH produced murideoxycholic acid (MDCA), isolithocholic acid (ILCA), and 3-keto-5beta-cholanic acid (3KCA) as major metabolites and 6-ketolithocholic acid and ursodeoxycholic acid as minor metabolites. Experiments with hepatic microsomes prepared from rats pretreated with P450 inducers and with inhibitory antibodies indicated that CYP2C and CYP3A enzymes contribute to microsomal MDCA formation. Results obtained with a panel of recombinant P450 enzymes and CYP2D6 antiserum showed that CYP2D1 can also catalyze MDCA formation. Similar experimental evidence revealed that formation of 3KCA was mediated primarily by CYP3A enzymes. ILCA formation appeared to be catalyzed by a distinct pathway mediated largely by microsomal non-P450 enzymes. Based on the results obtained using lithocholic acid and 3KCA as substrates, a mechanism for the formation of ILCA involving a geminal diol intermediate is outlined. In conclusion, lithocholic acid was extensively metabolized by multiple P450 enzymes with the predominant biotransformation pathway being hydroxylation at the 6beta-position. This study provides an insight into possible routes of detoxification of lithocholic acid.

Membrane transporters play a critical role in the absorption, distribution, and elimination of both endogenous substrates and xenobiotics. Defects in transporter function can lead to altered drug disposition including toxicity or loss of efficacy. Inflammation is one condition during which variable drug response has been demonstrated, and this can be attributed, at least in part, to changes in the expression of transporter genes. Thus, knowledge of the mechanisms behind transporter regulation can significantly contribute to our ability to predict variations in drug disposition among individuals and during inflammatory disease. The discovery of several xenobiotic-activated nuclear hormone receptors during the past decade including the pregnane X receptor, constitutive androstane receptor, and farnesoid X receptor has contributed greatly toward this endeavor. These receptors regulate the expression of transporters such as P-glycoprotein, MRP2, MRP3, BCRP, and OATP2 (Oatp1a1/OATP1B1), all of which undergo altered expression during an inflammatory response. Nuclear receptors may therefore play an important role in mediating this effect. This review presents what is currently known about the role of nuclear receptors in transporter regulation during inflammation. The use of this knowledge toward understanding interindividual variation in drug response and drug interactions during inflammation as well toward the development of therapeutics to treat transporter-related diseases will also be discussed.

Activation of the pregnane X receptor (PXR) has been shown to protect against cholestatic hepatotoxicity. As PXR alters the expression of numerous hepatic bile acid transporters, we sought to delineate their potential role in hepatoprotection.

Wild-type (PXR+/+) and PXR-null (PXR-/-) mice were fed a 1% cholic acid (CA) diet with or without the PXR activator, PCN. Liver function was assessed along with the corresponding changes in hepatic gene expression.

CA administration caused significant hepatotoxicity in PXR+/+ mice and was associated with induction of several FXR and PXR regulated genes, which encode for bile acid transport and metabolizing proteins. Compared to CA alone, co-administration of PCN to CA-fed PXR+/+ mice significantly decreased hepatotoxicity and was associated with induction of MRP3 mRNA as well as CYP3A11 mRNA and functional activity. Unexpectedly, PXR-/- mice, which expressed significantly higher basal and CA-induced levels of MRP2, MRP3, OSTalpha, OSTbeta, OATP2 and CYP3A11, were dramatically less sensitive to CA hepatotoxicity than PXR+/+ mice.

Protection of PXR+/+ mice against CA-induced hepatotoxicity by PCN is associated with the induction of MRP3 and CYP3A11 expression. Resistance against CA-induced hepatotoxicity in PXR-/- mice may result from higher basal and induced expression of bile acid transporters, particularly MRP3. These findings emphasize the importance of transport by MRP3 and metabolism as major protective pathways against cholestatic liver injury.

Reactive oxygen species (ROS), such as H2O2, are implicated in normal and pathological liver function. However, due to the lack of suitable in vivo models of ROS generation the (patho) physiological relevance of H2O2 remains controversial.

We established a novel model of sustained hepatic H2O2 release using intravenous administration of purified Aspergillus niger glucose oxidase (GOX) in rats.

GOX is rapidly cleared from the blood stream and almost exclusively localizes to Kupffer cells. GOX maintained its ability to generate H2O2 over 24h. While sublethal GOX doses induced hepatocellular necrosis, our morphological and functional studies show that lower levels of GOX which generate H2O2 comparable to release by inflammatory cells are non-toxic and do not induce histological inflammation. However, these non-toxic H2O2 levels increased oxidized glutathione and induced heme oxygenase 1 in the liver. In addition, several hepatocyte transporters were downregulated, while no decrease of bile formation, a sensitive marker of liver function, was observed.

Our in vivo model allows to study the effects of extracellular H2O2 in the liver as is released by inflammatory cells. Thus analysis of the role of this major ROS in the absence of confounding inflammatory cofactors will be possible.

It is hypothesized that during cholestasis, the liver, kidney, and intestine alter gene expression to prevent BA accumulation; enhance urinary excretion of BA; and decrease BA absorption, respectively. To test this hypothesis, mice were subjected to either sham or bile-duct ligation (BDL) surgery and liver, kidney, duodenum, ileum, and serum samples were collected at 1, 3, 7, and 14 days after surgery. Serum total BA concentrations were 1-5 mumol/l in sham-operated mice and were elevated at 1, 3, 7, and 14 days after BDL, respectively. BDL decreased liver Ntcp, Oatp1a1, 1a5, and 1b2 mRNA expression and increased Bsep, Oatp1a4, and Mrp1-5 mRNA levels. In kidney, BDL decreased Oatp1a1 and increased Mrp1-5 mRNA levels. In intestine, BDL increased Mrp3 and Ibat mRNA levels in ileum. BDL increased Mrp1, 3, 4, and 5 protein expression in mouse liver. These data indicate that the compensatory regulation of transporters in liver, kidney, and intestine is unable to fully compensate for the loss of hepatic BA excretion because serum BA concentration remained elevated after 14 days of BDL. Additionally, hepatic and renal Oatp and Mrp genes are regulated similarly during extrahepatic cholestasis, and may suggest that transporter expression is regulated not to remove bile constituents from the body, but instead to remove bile constituents from tissues.

Cholestasis results in intrahepatic accumulation of cytotoxic bile acids which cause liver injury ultimately leading to biliary fibrosis and cirrhosis. Cholestatic liver damage is counteracted by a variety of intrinsic hepatoprotective mechanisms. Such defense mechanisms include repression of hepatic bile acid uptake and de novo bile acid synthesis. Furthermore, phase I and II bile acid detoxification is induced rendering bile acids more hydrophilic. In addition to "orthograde" export via canalicular export systems, these compounds are also excreted via basolateral "alternative" export systems into the systemic circulation followed by renal elimination. Passive glomerular filtration of hydrophilic bile acids, active renal tubular secretion, and repression of tubular bile acid reabsorption facilitate renal bile acid elimination during cholestasis. The underlying molecular mechanisms are mediated mainly at a transcriptional level via a complex network involving nuclear receptors and other transcription factors. So far, the farnesoid X receptor FXR, pregnane X receptor PXR, and vitamin D receptor VDR have been identified as nuclear receptors for bile acids. However, the intrinsic adaptive response to bile acids cannot fully prevent liver injury in cholestasis. Therefore, additional therapeutic strategies such as targeted activation of nuclear receptors are needed to enhance the hepatic defense against toxic bile acids.

Pharmacokinetic studies of the drugs administered to subjects with mechanical cholestasis are scarce. The purpose of the present study was to examine the effects of bile duct ligation of 3 days (peak of elevation of serum bile acids and bilirubin) on the systemic and renal PAH clearance and on the expression of cortical renal OAT1 and OAT3 in a rat model. PAH is the prototypical substrate of the renal organic anion transport system. Male Wistar rats underwent a bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BDL rats displayed a significantly lower systemic PAH clearance. Renal studies revealed a reduction in the renal clearance and in the excreted and secreted load of PAH in BDL rats. The OAT1 protein expression in kidney homogenates was not modified, but it decreased in the basolateral membranes from BDL rats. In contrast, OAT3 abundance in both kidney cortex homogenates and in basolateral membranes increased by 3 days after the ligation. Immunocytochemical studies (light microscopic and confocal immunofluorescence microscopic analyses) confirmed the changes in the renal expression of these transport proteins. The present study demonstrates the key role of OAT1 expression in the impaired elimination of PAH after 3 days of obstructive cholestasis.

In juvenile rats born from mothers with obstructive cholestasis during pregnancy (OCP), transient latent cholestasis together with alterations in the secretion of biliary lipids have been reported. Here we investigated whether the expression of genes involved in this function is already modified at birth and examined the effect of treating pregnant rats with ursodeoxycholic acid (UDCA; i.g., 60 microg/100 g b.w./day). Cholanemia was markedly higher in mothers with OCP, and was further increased by UDCA. In the Control pups, cholanemia increased after birth, whereas in OCP and OCP+UDCA pups, hypercholanemia decreased after birth. Steady-state mRNA levels in neonatal liver were measured by real-time quantitative RT-PCR. The expression of basolateral bile acid transporters was not affected by OCP and was unchanged (Oatp1/1a1 and Oatp4/1b2) or moderately increased (Ntcp and Oatp2/1a4) by UDCA. In both groups, the expression of ABC proteins was either not modified (Bsep, Bcrp and Mrp2) or enhanced (Mrp1 and Mrp3), that of phospholipid flippase Mdr2 was not changed, whereas that of cholesterol transporter Abcg5/Abcg8 was impaired. The expression of the nuclear receptor FXR was not affected by OCP or UDCA, whereas that of SHP and key enzymes in bile acid synthesis (Cyp7a1, Cyp8b1 and Cyp27) was increased in both groups. In conclusion, OCP affects the expression in the neonatal liver of genes involved in hepatobiliary function, which cannot be prevented, at this stage, by treating pregnant rats with UDCA, even though this treatment has been found to partially restore normal lipid secretion later during post-natal development.

Sex differences in drug pharmacokinetics have been well recognized and gender has been considered a risk factor for adverse events to medications. The aim of this study was to investigate the effect of gender on the expression of hepatocellular transport proteins involved in uptake and secretion of organic anions in rat.

Expression of the rat liver organic anion transporting polypeptides (Oatps) and multidrug resistance proteins (Mrps) was analysed by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis and immunofluorescence microscopy in male and female rats. Regulation of these transport proteins in response to the steroid dehydroepiandrosterone (DHEA) was investigated.

In untreated rats, protein expression significantly differed between genders being higher (Mrp2, Mrp3), comparable [Oatp1a1 (Oatp1); Oatp1b2 (Oatp4)] or lower [Oatp1a4 (Oatp2)] in female than in male rat. DHEA treatment over 3 days (100 mg d(-1)) led to a further increase in Mrp3 expression only in female rats. Mrp2 expression was not influenced by DHEA treatment. Oatp1a1 and Oatp1b2 were significantly down-regulated after DHEA treatment in both male and female rats. In contrast, Oatp1a4 was down-regulated in male rats only.

In rat, liver transport proteins of the Oatp and Mrp family are expressed and regulated in a gender-specific manner according to sexual differences in the hepatic metabolism of steroids and drugs. These findings may partly explain the well-known sex differences in hepatic handling of organic anions.

The human apical sodium-dependent bile acid transporter (hASBT) represents a potential target for prodrug design to increase oral drug absorption. Unfortunately, available monolayer cell culture models do not reliably express hASBT, and nonpolarized cells only allow for uptake assessment, which limits prodrug development efforts. The objective of this study was to develop and characterize a stably transfected hASBT-MDCK cell line.

cDNA encoding hASBT was cloned into pcDNA3.1-V5-polyHis-B to generate an expression plasmid that was then transfected into MDCK-II cells. Clonal populations were chosen based on high hASBT activity and monolayer integrity. Western blot confirmed the expression of the recombinant hASBT; functionality was characterized using taurocholic acid.

In the selected clone, hASBT-mediated taurocholate permeability across hASBT-MDCK monolayers was almost 25-fold higher with sodium, than without sodium where hASBT is not functional. In the presence of sodium, taurocholate and mannitol permeabilities were 23.0x10(-6) cm/sec and 2.60x10(-6) cm/s, respectively, indicating high hASBT functionality and monolayer integrity. hASBT-MDCK monolayer properties were stable over 6 months and demonstrated low within-day variability. Taurocholate uptake and inhibition kinetic parameters from hASBT-MDCK were similar to those obtained from hASBT-COS7 model, confirming hASBT functionality in hASBT-MDCK.

Results indicate that the developed hASBT-MDCK system is a competent, high-expression, stable assay for hASBT transport and inhibition studies.

The farnesoid X receptor (FXR, NR1H4) regulates bile acid and lipid homeostasis by acting as an intracellular bile acid-sensing transcription factor, resulting in altered expression of enzymes and transporters involved in bile acid synthesis and transport. Here, we quantitatively analyzed the alterations in expression levels of drug transporters, mainly organic anion-transporting polypeptides (oatp), in wild-type and FXR-null mice to evaluate the role of FXR in their expression and regulation by cholic acid. Changes in the mRNA amounts in liver, kidney, small intestine, and testis in FXR-null mice fed with or without a supplement of 0.5% cholic acid in the diet were analyzed by semiquantitative RT-PCR. In FXR-null mice, the mRNA levels of oatp1, oatp2, oatp3, and octn1 were lower than those of wild-type mice in kidney and testis, while there was no difference in liver or small intestine. Cholic acid feeding led to significantly decreased levels of expression of oatp1 and oct1 and an increased level of expression of oatp2 in wild-type mouse liver. In FXR-null mice, oatp1 and other transporters were downregulated in liver, kidney, and testis, whereas small intestine ASBT, octn2, and pept1 were upregulated. Our results suggested that FXR is involved in the transcriptional regulation of oatp and other transporters in a tissue-specific manner. Furthermore, the effect of cholic acid treatment indicates the involvement of regulatory mechanism(s) other than FXR.

The increasing cost of drug development is partially due to our failure to identify undesirable compounds at an early enough stage of development. The application of higher throughput screening methods have resulted in the generation of very large datasets from cells in vitro or from in vivo experiments following the treatment with drugs or known toxins. In recent years the development of systems biology, databases and pathway software has enabled the analysis of the high-throughput data in the context of the whole cell. One of the latest technology paradigms to be applied alongside the existing in vitro and computational models for absorption, distribution, metabolism, excretion and toxicology (ADME/Tox) involves the integration of complex multidimensional datasets, termed toxicogenomics. The goal is to provide a more complete understanding of the effects a molecule might have on the entire biological system. However, due to the sheer complexity of this data it may be necessary to apply one or more different types of computational approaches that have as yet not been fully utilized in this field. The present review describes the data generated currently and introduces computational approaches as a component of ADME/Tox. These methods include network algorithms and manually curated databases of interactions that have been separately classified under systems biology methods. The integration of these disparate tools will result in systems-ADME/Tox and it is important to understand exactly what data resources and technologies are available and applicable. Examples of networks derived with important drug transporters and drug metabolizing enzymes are provided to demonstrate the network technologies.

Ursodeoxycholic acid (UDCA) is beneficial in cholestatic diseases but its molecular mechanisms of action remain to be clearly elucidated. Other bile acids, such as chenodeoxycholic (CDCA), are agonists for the nuclear farnesoid X receptor (FXR) and regulate the expression of genes relevant for bile acid and cholesterol homeostasis. In ileal cells CDCA, through the FXR, up-regulates the expression of the ileal bile acid-binding protein (IBABP), implicated in the enterohepatic circulation of bile acids. We report that UDCA (100 and 200 microM) induced a moderate increase of IBABP mRNA (approximately 10% of the effect elicited by 50 microM CDCA) in enterocyte-like Caco-2 cells and approximately halved the potent effect of CDCA (50 microM). On the contrary, UDCA reduced by 80-90% CDCA-induced IBABP transcription in hepatocarcinoma derived HepG2 cells. We confirmed that these effects on IBABP transcription required the FXR by employing a cell-based transactivation assay. Finally, in a receptor binding assay, we found that UDCA binds to FXR expressed in CHO-K1 cells (K(d)=37.7 microM). Thus, UDCA may regulate IBABP in Caco-2 cells, which express it constitutively, by acting as a partial agonist through a FXR mediated mechanism. The observation that in HepG2 cells, which do not express constitutively IBABP, UDCA was able to almost completely prevent CDCA-induced activation of IBABP promoter, suggests that tissue-specific factors, other than FXR, may be required for bile acid regulation of FXR target genes.

Ursodeoxycholic acid (UDCA) has beneficial effects in cholestatic liver diseases. In primary sclerosing cholangitis (PSC), there is evidence that high doses (+/- 20 mg/kg) of UDCA may be more effective than average doses. Biliary enrichment of UDCA at such high doses may represent the decisive factor for its beneficial effect. Up to now it is not clear how high-dose UDCA correlates with its biliary enrichment and whether bacterial degradation of large amounts of UDCA may lead to an increased bacterial formation of more toxic hydrophobic bile acids. We determined the biliary bile acid composition in 56 patients with PSC including 30 patients with repeat bile samples treated with various doses of UDCA. At a UDCA dose of 10-13 mg/kg/d (n = 18) biliary UDCA represented 43.1% + 0.3% (mean + SD) of total bile acids; at a UDCA dose of 14-17 mg/kg (n = 14), its biliary content increased to 46.9% + 0.3%, at 18-21 mg/kg (n = 34) to 55.9% + 0.2%, at 22-25 mg/kg (n = 12) to 58.6% + 2.3%, and at 26-32 mg/kg (n = 8) to 57.7% + 0.4%. During UDCA treatment, the biliary content of all other bile acids was unchanged or decreased. In conclusion, biliary enrichment of UDCA increases with increasing dose and reaches a plateau at 22-25 mg/kg. There was no increase of toxic hydrophobic bile acids. If biliary enrichment of UDCA represents the decisive factor for its clinical effect, it seems likely that UDCA doses of up to 22-25 mg/kg may be more effective than lower doses.