Variations in genes encoding canalicular transportes, for biliary lipids may affect concentrations of biliary lipids in bile and promote cholesterol crystallization and gallstone formation. In our study we investigated the contribution of heterozygosity for common variations considered either potentially pathogenic or susceptibility alleles for cholesterol cholelithiasis in adults (c.523A>G (p.Thr175Ala) and c.1954A>G (p.Arg652Gly) in ABCB4, c.1331T>C (p.Val444Ala) in ABCB11 and c.55 G>C (p.Asp19His) in ABCG8) to the aetiology of paediatric idiopathic gallstone disease. Genotyping was performed in 35 paediatric subjects with idiopathic gallstones with positive family history for gallstones and 150 population controls. The ABCB4 variant p.Thr175Ala was found only in the controls, not in the patients. The frequency of the remaining three variant alleles and the corresponding genotypes did not differ between patients and controls. We conclude that the studied common variations in genes encoding canalicular transporters known to contribute to genetic predisposition to cholesterol gallstones in adulthood do not contribute specifically to the aetiology of paediatric idiopathic gallstones.

BACKGROUND

The bile salt export pump (BSEP/ABCB11) is the primary transporter for the excretion of bile acids from hepatocytes into bile. In human, inhibition of BSEP by drugs has been related to drug-induced cholestasis and subsequent cytotoxic effects. The role of BSEP in canine and feline liver diseases has not been studied in detail, but the same mechanism of inhibition by drugs as in humans could play a role in veterinary medicine. The aim of this study was to investigate the functional characteristics of feline Bsep in comparison with canine and human Bsep/BSEP with respect to substrate affinities and inhibitory potential of model drugs. Orthologs of all three species were cloned and cell membrane vesicles overexpressing feline, canine and human Bsep/BSEP were prepared for functional analyses.

RESULTS

The cDNA sequences of the open reading frames of feline, canine and human Bsep/BSEP showed a high similarity between the species. Functional studies demonstrated for all species a tendency to a higher affinity of BSEP/Bsep for the conjugated bile acid taurocholic acid (TCA) than glycocholic acid (GCA), and a higher affinity for GCA than for the unconjugated cholic acid (CA). The inhibitory potency of the model inhibitors cyclosporine A, troglitazone and ketoconazole was characterized against TCA uptake into BSEP/Bsep containing membrane vesicles. All three substances potently inhibited TCA uptake without significant species differences.

CONCLUSIONS

Structure and functional characteristics of cat, dog and human Bsep/BSEP appeared to be very similar, indicating that the properties of this transporter have been highly preserved among the different species. Therefore, inhibition of BSEP by drugs could also be a mechanism in cholestasis and liver disease in veterinary relevant animal species. This model could be used to predict drug-induced liver injury caused by BSEP inhibition at an early stage in veterinary drug development.

The formation of cholesterol gallstones involves very complex imbalances, such as alterations in the secretion of biliary lipids (which involves the ABCG5, ABCG8, ABCB4 and ABCB11 transporters), biochemical and immunological reactions in the gallbladder that produce biliary sludge (mucins), physicochemical changes in the structure of cholesterol (crystallization), alterations in gallbladder motility, changes in the intestinal absorption of cholesterol (ABCG5/8 transporters and Niemann-Pick C1L1 protein) and alterations in small intestine motility. Some of these proteins have been studied at the clinical and experimental levels, but more research is required. In this review, we discuss the results of studies on some molecules involved in the pathophysiology of gallstones that may be future therapeutic targets to prevent the development of this disease, and possible sites for treatment based mainly on the absorption of intestinal cholesterol (Niemann-Pick C1L1 and ABCG5/8 proteins).

In the formation of cholesterol gallstones, cholesterol hypersecretion into bile causing cholesterol supersaturation and crystallization appears to be the primary factor, with disturbed gallbladder and intestinal motility as secondary factors. Although intestinal uptake mechanisms have not yet been fully elucidated, the HDL receptor scavenger receptor B1 (SRB1) may be involved. Since HDL-cholesterol, both from the intestine and peripheral sources, is the preferred type of cholesterol for biliary secretion, increased HDL transport to the liver can also cause cholesterol hypersecretion in bile. In the hepatocyte, bile formation is regulated by several transmembrane proteins, all belonging to the ABC family. A change in the activity in one of these proteins can have a profound impact on biliary lipid secretion. The bile salt export pump (BSEP or ABCB11) regulates the excretion of bile salts into bile and mutations cause severe cholestasis. The second ABC transporter, ABCB4 (MDR3) regulates the secretion in bile of phosphatidylcholine (PC), while ABCG5/G8 is active in the excretion of cholesterol and sterols into bile. These transporters also facilitate transport of sterols back into the intestinal lumen. Mutations in either of these genes cause sitosterolaemia with increased absorption of plant sterols and cholesterol. Until now, evidence for a genetic background of human gallstone disease is mostly indirect and based on ethnic differences. Only two single gene defects are associated with gallstones. One is an ABCB4 mutation which causes a deficiency in biliary PC secretion and the other is a CYP7A1 mutation, the rate-limiting enzyme in the synthesis of bile salts from cholesterol in the liver. Recently, several common DNA polymorphisms in the ABCG8 gene were discovered that are associated with variations in plasma sterols, which could also influence biliary cholesterol secretion, but there is still a paucity of human studies.

OBJECTIVE

To date, no studies concerning the presence of small intestinal bacterial overgrowth in patients with progressive familial intrahepatic cholestasis were published. Based upon characteristic of progressive familial intrahepatic cholestasis one can expect the coexistence of small intestinal bacterial overgrowth. The aim of the study was to assess the incidence of small intestinal bacterial overgrowth in patients with progressive familial intrahepatic cholestasis.

METHODS

26 patients aged 8 to 25 years with progressive familial intrahepatic cholestasis were included in the study. Molecular analysis of ABCB11 gene was performed in the vast majority of patients. In all patients Z-score for body weight and height, biochemical tests (bilirubin, bile acid concentration, fecal fat excretion) were assessed. In all patients hydrogen-methane breath test was performed.

RESULTS

On the basis of first hydrogen-methane breath test, diagnosis of small intestinal bacterial overgrowth was confirmed in 9 patients (35%), 5 patients (19%) had borderline results. The second breath test was performed in 10 patients: in 3 patients results were still positive and 2 patients had a borderline result. The third breath test was conducted in 2 patients and positive results were still observed. Statistical analysis did not reveal any significant correlations between clinical, biochemical and therapeutic parameters in patients with progressive familial intrahepatic cholestasis and coexistence of small intestinal bacterial overgrowth.

CONCLUSIONS

Our results suggest that small intestinal bacterial overgrowth is frequent in patients with progressive familial intrahepatic cholestasis. Moreover, it seems that this condition has the tendency to persist or recur, despite the treatment.

Triple therapy combining the protease inhibitor telaprevir with interferon-α and ribavirin is a promising new option for long-term treatment of hepatitis C. The interaction potential of telaprevir has not yet been fully elucidated. The in vitro potency of telaprevir to inhibit P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) was assessed and its substrate characteristics for P-gp, BCRP and the multidrug resistance-associated proteins (MRPs, ABCCs) 1-3 were evaluated. The inducing properties of telaprevir on important drug-metabolising enzymes and transporters were also assessed and its ability to activate the pregnane X receptor (PXR) was investigated. Using growth inhibition assays, it was confirmed that telaprevir is a substrate of P-gp and it was demonstrated for the first time that it is not transported by BCRP and MRPs. Telaprevir only moderately inhibited P-gp in the calcein assay and did not inhibit BCRP in the pheophorbide A assay. In LS180 cells, telaprevir strongly induced mRNA expression of ABCG2 (4.3-fold at 30 μmol/L) and weakly induced ABCB11, CYP2C19 and UGT1A3. In contrast, telaprevir had no significant influence on mRNA expression of CYP3A4, UGT1A9, ABCB1, ABCC2 and SLCO1B1. In a reporter gene assay, telaprevir did not activate PXR. Thus, it appears unlikely that telaprevir induces CYP3A4 and P-gp in vivo in such a way as to provoke clinically relevant drug interactions. From the numerous perpetrator characteristics, telaprevir's inhibitor properties, especially of CYP3A4 and P-gp, appear to be the most relevant mechanism for drug interactions. The clinical relevance of the strong inducing effects on ABCG2 requires proper assessment.

OBJECTIVE

To investigate the contribution of ABCB4 mutations to pediatric idiopathic gallstone disease and the potential of hormonal contraceptives to prompt clinical manifestations of multidrug resistance protein 3 deficiency.

METHODS

Mutational analysis of ABCB4, screening for copy number variations by multiplex ligation-dependent probe amplification, genotyping for low expression allele c.1331T>C of ABCB11 and genotyping for variation c.55G>C in ABCG8 previously associated with cholesterol gallstones in adults was performed in 35 pediatric subjects with idiopathic gallstones who fulfilled the clinical criteria for low phospholipid-associated cholelithiasis syndrome (LPAC, OMIM #600803) and in 5 young females with suspected LPAC and their families (5 probands, 15 additional family members). The probands came to medical attention for contraceptive-associated intrahepatic cholestasis.

RESULTS

A possibly pathogenic variant of ABCB4 was found only in one of the 35 pediatric subjects with idiopathic cholesterol gallstones whereas 15 members of the studied 5 LPAC kindreds were confirmed and another one was highly suspected to carry predictably pathogenic mutations in ABCB4. Among these 16, however, none developed gallstones in childhood. In 5 index patients, all young females carrying at least one pathogenic mutation in one allele of ABCB4, manifestation of LPAC as intrahepatic cholestasis with elevated serum activity of gamma-glutamyltransferase was induced by hormonal contraceptives. Variants ABCB11 c.1331T>C and ABCG8 c.55G>C were not significantly overrepresented in the 35 examined patients with suspect LPAC.

CONCLUSIONS

Clinical criteria for LPAC syndrome caused by mutations in ABCB4 cannot be applied to pediatric patients with idiopathic gallstones. Sexual immaturity even prevents manifestation of LPAC.

Familial intrahepatic cholestases (FICs) are a heterogeneous group of autosomal recessive disorders of childhood that disrupt bile formation and present with cholestasis of hepatocellular origin. Three distinct forms are described: FIC1 and FIC2, associated with low/normal GGT level in serum, which are caused by impaired bile salt secretion due to defects in ATP8B1 encoding the FIC1 protein and defects in ABCB11 encoding bile salt export pump protein, respectively; FIC3, linked to high GGT level, involves impaired biliary phospholipid secretion due to defects in ABCB4, encoding multidrug resistance 3 protein. Different mutations in these genes may cause either a progressive familial intrahepatic cholestasis (PFIC) or a benign recurrent intrahepatic cholestasis (BRIC). For the purposes of the present study we genotyped 27 children with intrahepatic cholestasis, diagnosed on either a clinical or histological basis. Two BRIC, 23 PFIC and 2 BRIC/PFIC were identified. Thirty-four different mutations were found of which 11 were novel. One was a 2Mb deletion (5'UTR- exon 18) in ATP8B1. In another case microsatellite analysis of chromosome 2, including ABCB11, showed uniparental disomy. Two cases were compound heterozygous for BRIC/PFIC2 mutations. Our results highlight the importance of the pathogenic role of novel mutations in the three genes and unusual modes of their transmission.

BACKGROUND

Dubin-Johnson syndrome and intrahepatic cholestasis of pregnancy are rare chronic liver disorders. Dubin-Johnson syndrome may manifest as conjugated hyperbilirubinemia, darkly pigmented liver, presence of abnormal pigment in the parenchyma of hepatocytes and abnormal distribution of the coproporphyrin isomers I and III in the urine. Intrahepatic cholestatic jaundice of pregnancy presents as pruritus, abnormal liver biochemistry and increased serum bile acids.

METHODS

A Sri Lankan girl presented with recurrent episodes of jaundice. She had conjugated hyperbilirubinaemia with diffuse, coarse brown pigments in the hepatocytes. Urine coproporphyrin examination suggested Dubin-Johnson syndrome. Genetic studies confirmed missense homozygous variant p.Trp709Arg in the ATP-binding cassette sub-family C member 2 gene ABCC2 that encodes the Multidrug resistance-associated protein 2 that causes Dubin-Johnson syndrome. The gene study of the mother revealed the same missense variant in ABCC2/MRP2 but with a heterozygous status, and in addition a homozygous missense variant p.Val444Ala in the ATP-binding cassette, sub-family B member 11 gene ABCB11 that encodes the bile salt export pump.

CONCLUSIONS

Dubin-Johnson syndrome should be considered when the common causes for conjugated hyperbilirubinaemia have been excluded, and patient has an increased percentage of direct bilirubin relative to total bilirubin concentration. Its early diagnosis prevents repeated hospital admissions and investigations. Knowledge of a well known homozygous variant in ABCB11 gene could help in the management of pregnancy.

OBJECTIVE

Tsumura-Suzuki obese diabetic (TSOD) is a good model of metabolic syndrome showing typical lesions found in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, and develops spontaneous hepatic tumors with a high frequency. Majority of the developing tumors overexpress glutamine synthetase (GS), which is used as a marker of hepatocellular carcinoma (HCC). The aim of this study is to assess the status of expression of metabolism-related genes and the level of bile acids in the TSOD mice-derived tumors and to determine the association with metabolic dysregulation between human HCC and TSOD mice-derived tumors.

METHODS

GS-positive hepatic tumors or adjacent normal tissues from 71-week-old male TSOD mice were subjected to immunohistochemical staining, quantitative RT-PCR (qRT-PCR), quantitation of cholic acid and taurocholic acid.

RESULTS

We found that downregulation of the rate-limiting enzyme for betaine synthesis (BADH), at both mRNA and protein levels in GS-positive TSOD mice-derived tumors. Furthermore, the bile acid receptor FXR and the bile acid excretion pump BSEP (Abcb11) were found to be downregulated, whereas BAAT and Akr1c14, involved in primary bile acid synthesis and bile acid conjugation, were found to be upregulated at mRNA level in GS-positive TSOD mice-derived tumors. BAAT and Akr1c14 were also overexpressed at protein levels. Total cholic acid was found to be increased in GS-positive TSOD mice-derived tumors.

CONCLUSIONS

Our results strongly support the significance of TSOD mice as a model of spontaneously developing HCC.

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

BACKGROUND

BSEP disease results from mutations in ABCB11, which encodes the bile salt export pump (BSEP). BSEP disease is associated with an increased risk of hepatobiliary cancer.

METHODS

A 36 year old woman with BSEP disease developed pancreatic adenocarcinoma at age 36. She had been treated with a biliary diversion at age 18. A 1.7 x 1.3 cm mass was detected in the pancreas on abdominal CT scan. A 2 cm mass lesion was found at the neck and proximal body of the pancreas. Pathology demonstrated a grade 2-3 adenocarcinoma with invasion into the peripancreatic fat.

CONCLUSIONS

Clinicians should be aware of the possibility of pancreatic adenocarcinoma in patients with BSEP disease.

The occurrence of cholestasis during pregnancy may be due to several disorders. These include pregnancyspecific diseases, like intrahepatic cholestasis of pregnancy(ICP), as well as to other causes such as oligosymptomatic choledocolitiasis, viral hepatitis and other underlying liver disorders like primary biliary cirrhosis. In recent years, the discovery of mutations in hepatobiliary transporters genes responsible of some rare forms of genetic cholestasis have led to the study of this mutations in pregnant women with cholestasis. Thus, mutations in the hepatic phospholipid transporter(MDR3, ABCB4), in the aminophospholipid transporter ATP8B1 and in the bile salt export pump (BSEP, ABCB11) have been found in patients diagnosed as ICP. However, patients included in these studies belong to a heterogeneous population, which may not represent true cases of ICP since some reports include patients diagnosed in the first trimester of pregnancy, with elevated serum levels of gamma-glutamyl transpeptidase and clear evidence of chronic liver disease. Thus, consideration must be given to the possibility of other rare underlying hepatic disorders may be unmasked during pregnancy with cholestasis as its first manifestation.

We report the case of a 40-years-old female patient with recurrent cholestatic liver disease who presented twice with severe intrahepatic cholestasis of pregnancy and pronounced choledocholithiasis between pregnancies. Bile duct stones were removed endoscopically and a laparoscopic cholecystectomy was performed after the second pregnancy. Liver histology revealed intrahepatic cholestasis with portal inflammation and fibrosis, resembling progressive familial intrahepatic cholestasis (PFIC). Molecular genetic studies identified the heterozygous mutation c.957C>> T in the ABCB4 gene encoding the hepatobiliary phospholipid transporter. This is the first report of this mutation that introduces a stop codon in an index patient with intrahepatic cholestasis of pregnancy and multiple bile duct stones. In addition, we detected the ABCB11 polymorphism V 444A, which is associated with a decreased expression of the bile salt export pump. Whereas homozygous carriers of the ABCB4 mutation develop PFIC type 3, the heterozygous ABC transporter mutations represent genetic risk factors for cholelithiasis and recurrent cholestatic hepatitis upon challenge with oral contraceptives or during pregnancy. Of note, the patient presented with normal serum gamma-glutamyltranspeptidase activities during pregnancy-associated cholestatic episodes but normal liver enzymes after delivery, whereas choledocholithiasis was associated with high gamma-glutamyl transpeptidase levels. It is unknown whether ursodeoxycholic acid prevents cholestasis or gallstones in patients with ABCB4 deficiency.

OBJECTIVE

Cholestasis promotes endoplasmic reticulum (ER) stress in the liver, however, the effect of ER stress on hepatic bile acid metabolism is unknown. We aim to determine the effect of ER stress on hepatic bile acid synthesis and transport in mice.

METHODS

ER stress was induced pharmacologically in C57BL/6J mice and human hepatoma (HepG2) cells. The hepatic expression of genes controlling bile acid synthesis and transport was determined. To measure the activity of the primary bile acid synthetic pathway, the concentration of 7α-hydroxy-4-cholesten-3-1 was measured in plasma.

RESULTS

Induction of ER stress in mice and HepG2 cells rapidly suppressed the hepatic expression of the primary bile acid synthetic enzyme, cholesterol 7α-hydroxylase. Plasma levels of 7α-hydroxy-4-cholesten-3-1 were reduced in mice subjected to ER stress, indicating impaired bile acid synthesis. Induction of ER stress in mice and HepG2 cells increased expression of the bile salt export pump (adenosine triphosphate binding cassette [Abc]b11) and a bile salt efflux pump (Abcc3). The observed regulation of Cyp7a1, Abcb11, and Abcc3 occurred in the absence of hepatic inflammatory cytokine activation and was not dependent on activation of hepatic small heterodimer partner or intestinal fibroblast growth factor 15. Consistent with suppressed bile acid synthesis and enhanced bile acid export from hepatocytes, prolonged ER stress decreased the hepatic bile acid content in mice.

CONCLUSIONS

Induction of ER stress in mice suppresses bile acid synthesis and enhances bile acid removal from hepatocytes independently of established bile acid regulatory pathways. These data show a novel function of the ER stress response in regulating bile acid metabolism.

Background: Intrahepatic cholestasis of pregnancy (ICP) is characterized by pruritus and cholestasis in late pregnancy and results in adverse pregnancy outcomes, including preterm delivery and birth weight, which are affected by the genetic and environmental background. However, until now, the genetic architecture of ICP has remained largely unclear.

Methods: Twenty-six clinical data points were recorded for 151 Chinese ICP patients. The data generated from whole-exome sequencing (WES) using the BGISEQ-500 platform were further analyzed by Burrows-Wheeler Aligner (BWA) software, Genome Analysis Toolkit (GATK), ANNOVAR tool, etc. R packages were used to conduct t-test, Fisher's test and receiver operating characteristic (ROC) curve analyses.

Results: We identified eighteen possible pathogenic loci associated with ICP disease in known genes, covering ABCB4, ABCB11, ATP8B1 and TJP2. The loci Lys386Gln, Gly527Gln and Trp708Ter in ABCB4, Leu589Met, Gln605Pro and Gln1194Ter in ABCB11, and Arg189Ser in TJP2 were novel discoveries. In addition, WES analysis indicated that the gene ANO8 involved in the transport of bile salts is newly identified as associated with ICP. The functional network of the ANO8 gene confirmed this finding. ANO8 contained 8 rare missense mutations that were found in eight patients among the 151 cases and were absent from 1029 controls. Out of the eight SNPs, 3 were known, and the remaining five are newly identified. These variants have a low frequency, ranging from 0.000008 to 0.00001 in the ExAC, gnomAD - Genomes and TOPMED databases. Bioinformatics analysis showed that the sites and their corresponding amino acids were both highly conserved among vertebrates. Moreover, the influences of all the mutations on protein function were predicted to be damaging by the SIFT tool. Combining clinical data, it was found that the mutation group (93.36 µmol/L) had significantly (P = 0.038) higher total bile acid (TBA) levels than the wild-type group (40.81 µmol/L).

Conclusions: To the best of our knowledge, this is the first study to employ WES technology to detect genetic loci for ICP. Our results provide new insights into the genetic basis of ICP and will benefit the final identification of the underlying mutations.

Keywords: ANO8; Intrahepatic cholestasis of pregnancy; Mutations; Whole-exome sequencing.

Clinical and epidemiological findings implicate genetic predisposition and the effects of elevated steroids in pregnancy in the pathogenesis of intrahepatic cholestasis of pregnancy. To date, a number of studies have identified polymorphisms encoding biliary canalicular transporters, including those encoded by ABCB4 and ABCB11, which are associated with intrahepatic cholestasis of pregnancy. Questions remain regarding divergent findings between populations and the relative contributions of these polymorphisms. In a large study of Western European women with intrahepatic cholestasis of pregnancy, Dixon et al. (this issue) provide further insights into the genetics of this cholestatic syndrome, which contribute to ongoing evaluation of cholestasis generally.

Bile acid imbalance causes progressive familial intrahepatic cholestasis type 2 (PFIC2) or type 3 (PFIC3) - severe liver diseases associated with genetic defects in the biliary bile acid transporter BSEP (ABCB11), or phosphatidylcholine transporter MDR3 (ABCB4), respectively. Mdr2 -/- mice (a PFIC3 model) develop progressive cholangitis, ductular proliferation, periportal fibrosis, and hepatocellular carcinoma (HCC) because the non-micelle-bound bile acids in the bile of these mice are toxic. We asked whether the highly hydrophilic bile acids generated by Bsep -/- mice could protect Mdr2 -/- mice from progressive liver damage. We generated double knockout (DKO: Bsep -/- and Mdr2 -/-) mice. Their bile acid composition resembles that of Bsep-/- mice, with increased hydrophilic muricholic acids, tetrahydroxylated bile acids (THBA), and reduced hydrophobic cholic acid. These mice lack the liver pathology of their Mdr2 -/- littermates. The livers of DKO mice have gene expression profiles very similar to Bsep -/- mice, with 4410 out of 6134 gene expression changes associated with the Mdr2 -/- mutation being suppressed. Feeding with THBA partially alleviates liver damage in the Mdr2 -/- mice. Conclusion: Hydrophilic changes to biliary bile acid composition, including introduction of THBA, can prevent the progressive liver pathology associated with the Mdr2 -/- (PFIC3) mutation.

Delamanid (Deltyba, OPC-67683) is the first approved drug in a novel class of nitro-dihydro-imidazooxazoles developed for the treatment of multidrug-resistant tuberculosis. Patients with tuberculosis require treatment with multiple drugs, several of which have known drug-drug interactions. Transporters regulate drug absorption, distribution, and excretion; therefore, the inhibition of transport by one agent may alter the pharmacokinetics of another, leading to unexpected adverse events. Therefore, it is important to understand how delamanid affects transport activity. In the present study, the potencies of delamanid and its main metabolites as the substrates and inhibitors of various transporters were evaluated in vitro Delamanid was not transported by the efflux ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), solute carrier (SLC) transporters, organic anion-transporting polypeptides, or organic cation transporter 1. Similarly, metabolite 1 (M1) was not a substrate for any of these transporters except P-gp. Delamanid showed no inhibitory effect on ABC transporters MDR1, BCRP, and bile salt export pump (BSEP; ABCB11), SLC transporters, or organic anion transporters. M1 and M2 inhibited P-gp- and BCRP-mediated transport but did so only at the 50% inhibitory concentrations (M1, 4.65 and 5.71 μmol/liter, respectively; M2, 7.80 and 6.02 μmol/liter, respectively), well above the corresponding maximum concentration in plasma values observed following the administration of multiple doses in clinical trials. M3 and M4 did not affect the activities of any of the transporters tested. These in vitro data suggest that delamanid is unlikely to have clinically relevant interactions with drugs for which absorption and disposition are mediated by this group of transporters.

OBJECTIVE

Metabolism and transport play major roles in life-long exposure to endogenous and exogenous carcinogens. We therefore explored associations between polymorphisms in absorption, distribution, metabolism and elimination genes and the risk and prognosis of castration-resistant prostate cancer (CRPC).

METHODS

A total of 634 genotypes were tested in 74 patients using the Affymetrix DMETv1.0 platform.

RESULTS

No relation to risk was found. Three SNPs were associated with CRPC prognosis in Caucasians: ABCB11 rs7602171G>A (p = 0.003; n = 30; hazard ratio [HR]: 0.307), GSTP1 rs1799811C>T (p = 0.001; n = 38; HR: 0.254) and SLC5A6 rs1395 (p = 0.004; n = 35; HR: 3.15). Two other polymorphisms among Caucasians were associated with interesting trends: ABCB4 rs2302387C>T (p = 0.039) and ABCC5 rs939339A>G (p = 0.018).

CONCLUSIONS

This exploratory study is the first to show that polymorphisms in several absorption, distribution, metabolism and elimination genes may be associated with CRPC prognosis.

OBJECTIVE

Fatty liver alters liver transporter expression. Caloric restriction (CR), the recommended therapy to reverse fatty liver, increases Sirtuin1 deacetylase activity in liver. This study evaluated whether CR and CR mimetics reversed obesity-induced transporter expression in liver and hepatocytes.

METHODS

mRNA and protein expression was determined in adult lean (lean) and leptin-deficient obese (OB) mice fed ad libitum or placed on 40% (kCal) reduced diet. Hepatocytes were isolated from lean and OB mice, treated with AMP Kinase activators, and gene expression was determined.

RESULTS

CR decreased Oatp1a1, Oatp1b2, and Abcb11 mRNA expression in lean, but not OB mice. CR increased Abcc2 mRNA OB livers, whereas protein expression increased in both genotypes. CR increased Abcc3 protein expression increased in OB livers. CR did not alter Abcc1, 4 and 5 mRNA expression in lean mice but decreased expression in livers of OB mice. CR increased Abcc4 protein in lean, but not OB mice.

CONCLUSIONS

CR restriction reversed the expression of some, but not all transporters in livers of OB mice. Overall, these data indicate a potential for CR to restore some hepatic transporter changes in OB mice, but suggest a functional leptin axis is needed for reversal of expression for some transporters.

The cholesterol-lowering effects of tomato pomace (TP), tomato seed oil (TSO) and defatted tomato seed (DTS) were determined in male Golden Syrian hamsters. Hamsters fed high-fat diets containing 10% TSO or 18% DTS were compared to a diet containing 10% corn oil and 10% microcrystalline cellulose (control 1), 42% TP were compared to 25% microcrystalline cellulose (control 2). TP, TSO and DTS reduced hepatic total cholesterol (TC) content. DTS also lowered plasma TC and low-density lipoprotein cholesterol (LDL-C) concentrations. Fecal excretion of lipid, bile acid and cholesterol increased in the DTS group compared to control 1. DTS-fed hamsters had higher levels of hepatic CYP7A1, CYP51, ABCB11, and ABCG5 gene expression than control, suggesting both hepatic bile acid and cholesterol synthesis increased due to increased fecal excretion of bile acid and cholesterol. The results suggest that protein, dietary fibre or phenolic compounds in DTS may be responsible for plasma cholesterol decrease.

European studies have revealed that the ABCB11 c.1331T>C (V444A) polymorphism (rs2287622) C-allele frequency is higher among patients with drug-induced cholestasis. Given the low incidence of this disease, however, this association has not been sufficiently elucidated. We aimed to investigate the significance of this polymorphism in Japanese patients. We determined ABCB11 V444A polymorphism frequencies and HLA genotypes in two independent drug-induced cholestasis cohorts. Expression and taurocholate transport activity of proteins from 444A variants were analyzed using Madin-Darby canine kidney II cells. In cohort 1 (n = 40), the V444A polymorphism C-allele frequency (66%) was lower than that in controls (n = 190, 78%), but this difference was not significant (P = 0.09). In cohort 2 (n = 119), comprising patients with cholestatic (n = 19), hepatocellular (n = 74), and mixed (n = 26) liver injuries, the C-allele frequency was lower among patients with cholestatic liver injury (68%) than among those with hepatocellular (75%) or mixed liver injury (83%), although this difference was not significant. In cohort 1, HLA-A*0201 was observed more frequently in patients (22%) than in controls [11%; P = 0.003; odds ratio, 2.4 (95% confidence interval, 1.4-4.0)]. Taurocholate transport activity of 444A-encoded protein was significantly lower than that of 444V-encoded protein (81% of 444V, P < 0.05) because of the reduced protein stability. In conclusion, ABCB11 444A had slightly reduced transport activity, but it did not contribute to the occurrence of drug-induced cholestasis in Japanese patients. Therefore, genetic susceptibility to acquired cholestasis may differ considerably by ethnicity.