Chronic hepatitis C (CHC)-related mortality generally results from cirrhosis and subsequent complications. We aimed to investigate the potential role of plasma bile acid levels and ABCB11 1331T>> C (V444A, rs2287622) (ATP-binding cassette subfamily B, member 11) gene polymorphism in fibrosis prediction in CHC genotype 4 patients.

This case control study included 85 healthy control and the following 225 subjects: 170 adult patients infected with hepatitis C virus (HCV) and categorized into three groups according to liver biopsy; no fibrosis group (F0) (n=33), early fibrosis group (F1-F2) (n=61), and advanced fibrosis group (F3-F4) (n=76). Fasting bile acid levels, hepatitis C virus (HCV) genotyping, and ABCB11 1331T>> C gene polymorphism were assessed.

The frequency of the variant homozygote genotype CC in advanced fibrosis was significantly higher than that in early fibrosis (48.7% vs. 36.1%) (odd ratio, OR =2.58; 95% confidence interval, CI=1.07-6.20; p=0.03). C allele was significantly represented in advanced fibrosis (65.8%) compared with that in early fibrosis (51.6%) (OR=1.80, 95% CI=1.10-2.93, p=0.01). A significant elevation of plasma bile acid levels in advanced fibrosis was observed compared with those in early fibrosis (p≤0.001). Receiver operating characteristic curve for plasma bile acid levels at cutoff value of 75.5 μmol/L had a 59% specificity and 97.4% sensitivity as a predictor of advanced hepatic fibrosis (AUROC=0.78%).

We concluded that Egyptian patients having chronic hepatitis C genotype 4 with CC genotype of ABCB11 SNP 1331T>> C and high plasma bile acid levels at cutoff value of 75.5 μmol/L were associated with advanced hepatic fibrosis.

Carbon monoxide (CO), a product of heme oxygenase (HMOX), has many beneficial biological functions and is a promising therapeutic agent for many pathological conditions. However, the kinetics of inhaled CO and its protective role in endotoxin-induced cholestasis is not fully known. Thus, our objective was to characterize the kinetics of inhaled CO and then investigate its use in early phase experimental endotoxin-induced cholestasis. Female Wistar rats were randomly divided into 4 groups: CON (control), LPS (lipopolysaccharide, 6 mg/kg), CO (250 ppm COx1h), and CO + LPS. Rats were sacrificed at 0-12 h after LPS administration. Tissues and blood were collected for liver injury markers and tissue CO distribution measurements. Livers were harvested for measurements of Hmox activity, Hmox1 mRNA expression, cytokines (IL10, IL6, TNF), and bile lipid and pigment transporters. Half-lives of CO in spleen, blood, heart, brain, kidney, liver, and lungs were 2.4 ± 1.5, 2.3 ± 0.8, 1.8 ± 1.6, 1.5 ± 1.2, 1.1 ± 1.1, 0.6 ± 0.3, 0.6 ± 0.2 h, respectively. CO treatment increased liver IL10 mRNA and decreased TNF expression 1 h after LPS treatment and prevented the down-regulation of bile acid and bilirubin hepatic transporters (Slc10a1, Abcb11, and Abcc2, p < 0.05), an effect closely related to the kinetics. The protective effect of CO against cholestatic liver injury persisted even 12 h after CO exposure, as shown by attenuation of serum cholestatic markers in CO-treated animals. CO exposure substantially attenuated endotoxin-induced cholestatic liver injury and was directly related to the kinetics of inhaled CO. This data underscores the importance of the kinetics of inhaled CO for the proper design of experimental and clinical studies of using CO as a treatment strategy.

OBJECTIVE

We evaluated the prevalence of low phospholipid-associated cholelithiasis, a specific form of cholelithiasis associated with at least 2 of the 3 following criteria: first symptoms before the age of 40; intrahepatic comet tail artefacts, sludge or microlithiasis on ultrasound imaging; and recurrence of symptoms after cholecystectomy.

METHODS

We prospectively studied the cases of 60 consecutive female patients under 30 with symptomatic cholelithiasis.

RESULTS

A diagnosis of low phospholipid-associated cholelithiasis was made in 14/60 patients (23%). The molecular analysis showed ABCB4 (n=4) and ABCB11 (n=4) gene mutations. Low phospholipid-associated cholelithiasis was frequently observed in non-overweight patients [13/27 (48%)], was present in most patients whose biliary symptoms occurred before the age of 18 [7/10 (70%)] and was often associated with cholangitis or acute pancreatitis [9/14 (64%), p<0.05] while "common" cholelithiasis was mainly associated with cholecystitis [16/46 (35%), p<0.05].

CONCLUSIONS

Nearly one quarter of the female patients under the age of 30 admitted for symptomatic cholelithiasis had low phospholipid-associated cholelithiasis; particularly if body weight was normal, the symptoms began before the age of 18 or in the presence of severe biliary complications.

Drug-induced intrahepatic cholestasis is one of the major causes of hepatotoxicity, which often occur during the drug discovery and development process. Human ATP-binding cassette transporter ABCB11 (sister of P-glycoprotein/bile salt export pump) mediates the elimination of cytotoxic bile salts from liver cells to bile, and, therefore, plays a critical role in the generation of bile flow. The authors have recently developed in vitro high-speed screening and quantitative structure-activity relationship analysis methods to investigate the interaction of ABCB11 with a variety of compounds. Based on the extent of inhibition of the bile salt export pump, the authors analysed the quantitative structure-activity relationship to identify chemical groups closely associated with the inhibition of ABCB11. This approach provides a new tool to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.

C57L/J (gallstone-susceptible) and AKR/J (gallstone-resistant) mice have been utilized for quantitative trait loci (QTL) analysis to identify the Lith 1 locus for cholelithiasis. Abcb11 encodes for the liver canalicular membrane bile salt export pump (BSEP), which maps to this QTL and is a candidate gene for Lith 1. We investigated the transmembrane transport of taurocholate in canalicular liver membrane vesicles isolated from these murine strains. Canalicular liver plasma membranes (cLPM) and RNA were isolated from C57L/J and AKR/J mice livers, and were utilized for Northern and Western blot analysis and functional (3)H-taurocholate uptake studies. ATP-dependent (3)H-taurocholate uptake was significantly higher in AKR/J, compared to C57L/J mice. V(max) was 127 vs. 42 pmol TC/mg/s in the murine strains, respectively, while K(m) was unchanged. In contrast, gene and protein expression of hepatic Abcb11 was increased three-fold in C57L/J, compared to AKR/J mice. Thus, Abcb11 bile salt transport activity per unit protein was reduced nine-fold in the C57L/J, compared to AKR/J mice. In contrast, canalicular membrane cholesterol:phospholipid content was also significantly higher in the C57L/J mice. We conclude that gallstone-susceptible C57L/J mice demonstrate increased gene and canalicular membrane expression of Abcb11, however, taurocholate transport is functionally diminished. The latter may be due to the increased cholesterol membrane content of the cLPM in C57L/J mice. These findings may be important for the pathogenesis of gallstone formation.

Intrahepatic cholestasis of pregnancy (ICP) is characterized by the occurrence of pruritus mostly in the third trimenon. Diagnosis is based on the presence of pruritus and elevated levels of serum bile acids in the absence of pruritic skin diseases. There is strong evidence of a genetic predisposition for ICP. Numerous studies have investigated the association of known cholestasis genes such as ABCB4 (also designated MDR3), ABCB11 ( BSEP) and ATP8B1 ( FIC1) with ICP. The results of these studies implicate a heterogeneous etiology of this syndrome. ICP increases the risk of preterm delivery and fetal loss. Furthermore, intense pruritus may necessitate premature induction of labor with its known higher frequency of complications for mother and child. Therefore, ICP pregnancies should be managed as high-risk pregnancies. Pharmaceuticals to alleviate pruritus or improve cholestasis like antihistamines, phenobarbital, anion exchange resins, dexamethasone or S-adenosylmethionine are not widely accepted because of questionable efficacy or side effects. Recent randomized studies have shown beneficial effects of ursodeoxycholic acid (UDCA) on laboratory data and pruritus in patients with ICP. Improved knowledge about the diagnostic classification of different types and pathophysiological mechanisms of ICP may allow for a more targeted treatment of this disease in future.

Bile salt export pump (BSEP) plays an important role in hepatic secretion of bile acids and its deficiency results in severe cholestasis and liver failure. Mutation of the ABCB11 gene encoding BSEP induces BSEP deficiency and progressive familial intrahepatic cholestasis type 2 (PFIC2). Because liver transplantation remains standard treatment for PFIC2, the development of a novel therapeutic option is desired. However, a well reproducible model, which is essential for the new drug development for PFIC2, has not been established. Therefore, we attempted to establish a PFIC2 model by using iPSC technology. Human iPSCs were generated from patients with BSEP-deficiency (BD-iPSC), and were differentiated into hepatocyte-like cells (HLCs). In the BD-iPSC derived HLCs (BD-HLCs), BSEP was not expressed on the cell surface and the biliary excretion capacity was significantly impaired. We also identified a novel mutation in the 5'-untranslated region of the ABCB11 gene that led to aberrant RNA splicing in BD-HLCs. Furthermore, to evaluate the drug efficacy, BD-HLCs were treated with 4-phenylbutyrate (4PBA). The membrane BSEP expression level and the biliary excretion capacity in BD-HLCs were rescued by 4PBA treatment. In summary, we succeeded in establishing a PFIC2 model, which may be useful for its pathophysiological analysis and drug development.

The aim of this study was to evaluate the response of ABC transporters, CYP1A and class alpha (α) GST genes, upon water and dietary exposures to benzo(a)pyrene (BaP) in Oreochromis niloticus. Partial mRNA sequences of ABC transporters (ABCB1b, ABCB11, ABCC1, ABCC2 and ABCG2) were identified, and their tissue distribution patterns evaluated in liver, gill and intestine, showing similarities with other fish and mammals. After 14 days of water exposure to BaP, ABC transporters mRNA expression was up-regulated, namely ABCC2 in gill (up to 16-fold) and ABCG2 in liver (up to 2-fold) and proximal intestine (up to 7-fold). CYP1A mRNA expression was up-regulated in water exposed animals, with maximum fold inductions of 5, 35 and 155, respectively in liver, gill and proximal intestine. After dietary exposure, intestinal CYP1A mRNA showed a 13-fold increase in exposed animals. No significant changes were seen in ABCB1b, ABCC1 and GSTα mRNA expression after both routes of exposure to BaP. In conclusion, this study has shown that transcriptional expression of some ABC transporters and CYP1A respond to the presence of BaP, indicating a possible involvement and cooperation in the detoxification process in Nile tilapia.

The bile salt export pump, encoded by ABCB11, is the predominant canalicular transport protein for biliary bile acid secretion. The level of ABCB11 expression in humans is widely variable yet the impact of this variability on human disease is not well defined. We aim to determine the effect of hepatic Abcb11 overexpression on the enterohepatic circulation (EHC) in mice. We used a stable isotope dilution technique in transgenic mice overexpressing hepatic Abcb11 (TTR-Abcb11) to determine the pool size, fractional turnover rate (FTR), and synthesis rate of the primary bile acid, cholic acid (CA). The gallbladder was cannulated to determine bile flow, bile acid composition, and the biliary secretion rates of CA, total bile acids, phospholipid, and cholesterol. The combined data allowed for estimation of the CA cycling time and the fraction of CA lost per cycle. Hepatic and intestinal gene and protein expression were determined by qPCR and Western blot. Abcb11 overexpression strongly decreased FTR and synthesis rate of CA. Abcb11 overexpression decreased the fraction of CA that was lost per cycle of the EHC. Hepatic expression of Cyp7a1 was suppressed by nearly 50% and ileal expression of FGF15 was increased more than eightfold in TTR-Abcb11 mice. Despite the increased intestinal reabsorption of bile acids, ileal Asbt expression was suppressed. Hepatic Abcb11 overexpression in mice increases the conservation of bile acids within the enterohepatic circulation. These data provide strong evidence for the existence of feed-forward communication between hepatic expression of a bile acid transport protein and the intestine.

Hepatitis A virus (HAV) infection resolves in most patients uneventfully within weeks from the onset of the disease. In rare cases, however, it may relapse or cause prolonged cholestasis. Here we present a case of a 36-year-old female patient who developed severe pruritus and jaundice three weeks after initially uncomplicated hepatitis A. A relapse of the infection was excluded. Since therapy with colestyramin, antihistaminics, naloxon and ursodeoxycholic acid (UDCA) did not improve symptoms, we decided to perform plasma absorption and to start rifampicin therapy. Under these measures, pruritus and jaundice, as well as serum bilirubin levels improved gradually and after four plasmapheresis sessions we were able to discharge the patient. Genetic testing showed the presence of two procholestatic polymorphisms, the c.3084 [GG] variant within the gene encoding the hepatocanalicular bile salt transporter ABCB11 and the c.711 [AT] variant of the phosphatidylcholine floppase ABCB4. We speculate that this compound ABCB4-ABCB11 genotype led to a severe intrahepatic cholestasis in the setting of HAV infection. In conclusion, our case suggests that polymorphisms within the hepatocanalicular transporters may contribute to a more pronounced course of HAV infection. Although dedicated studies in large cohorts of patients are needed to confirm this observation, we speculate that patients carrying procholestatic hepatobiliary transporter variants may benefit from vaccination against hepatitis A.

We report here a comprehensive evaluation of the effects of culture duration on the gene expression of P450 isoforms, uptake transporters and efflux transporters in human hepatocyte cultured in the absence and presence of the prototypical proinflammatory cytokine, interleukin-6 (IL-6). Primary collagen-matrigel sandwich cultures of human hepatocytes were cultured in supplemented William's E medium containing 0, 0.1, 0.5 and 5 ng/mL of IL-6 for the time periods of 2, 6, 12, 24 and 48 hrs. Real-time PCR was performed to quantify gene expression of acute phase proteins (suppressor of cytokine signaling 3 (SOCS-3), c-reactive protein (CRP) and lipopolysaccharide (LPS)-binding proteins (LBP)); P450 isoforms (CYPs 1A2, 2B6, 2C8, 2C9, 2D6, 3A4, and 3A5), uptake transporters (SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, SLCO2B1) and efflux transporters (ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2). SOCS-3, CRP, and LBP were extensively induced by IL-6, with maximal induction observed at 2 (SOCS-3) and 12 hrs (CRP; LBP), demonstrating that the cultured human hepatocytes responded to IL-6 treatment. In the untreated group (control), gene expression of P450 isoforms and uptake transporters decreased while efflux transporters remained relatively stable or increased with cultured duration. IL-6 predominantly caused down regulations of the genes studied, with the most significant changes observed at different treatment durations, apparently related to the stability of the basal levels of gene expression. For instance, for genes with unstable expression, which would decrease rapidly in culture (e.g CYP3A4), the most definitive down regulatory effects were observed at a relatively early time point (e.g. 12 hrs). In contrast, a longer treatment duration (e.g. 48 hrs) was required for genes with relatively stable expression levels in culture (e.g. ABCB1). Based on our findings, evaluation of multiple treatment durations rather than single treatment duration is recommended for the evaluation of biotherapeutics in cultured human hepatocytes where down regulation is expected.

BACKGROUND

Intrahepatic cholestasis of pregnancy (ICP) is a cholestasis condition caused by elevated levels of serum bile acids that mainly occurs in the third trimester of pregnancy. Maternal symptoms include pruritus; elevation of transaminases, biliary enzymes, and bilirubin levels; and abnormal liver function tests. Fetal symptoms include spontaneous preterm labor, fetal distress, and intrauterine death. It is more prevalent in the Caucasians and is rarely found in Asian countries, including Japan. The etiology of ICP has been reported as involving various factors such as, environmental factors, hormone balance, and genetic components. The genetic factors include single-nucleotide polymorphisms (SNPs) in the genes of canalicular transporters, including ABCB4 and ABCB11. It has also been reported that the combination of these SNPs induces severe cholestasis and liver dysfunction.

METHODS

Here, we report for the first time a 24-year Japanese case of severe ICP diagnosed by typical symptoms, serum biochemical analysis, and treated with the administration of ursodeoxycholic acid which improved cholestasis and liver injury and prevented fetal death. The sequence analysis showed SNPs reported their association with ICP in the ABCB11 (rs2287622, V444A) and ABCB4 (rs1202283, N168N) loci.

CONCLUSIONS

The risk of ICP has been reported to be population-specific, and it is rare in the Japanese population. Our case was successfully treated with ursodeoxycholic acid and the genetic sequence analysis has supported the diagnosis. Because genetic variation in ABCB4 and ABCB11 has also been reported in the Japanese population, we need to be aware of potential ICP cases in pregnant Japanese women although further studies are necessary.

UNASSIGNED

Marker detection is an important task in complex disease studies. Here we provide an association rule mining (ARM) based approach for identifying integrated markers through mutual information (MI) based statistically significant feature extraction, and apply it to acute myeloid leukemia (AML) and prostate carcinoma (PC) gene expression and methylation profiles.

UNASSIGNED

We first collect the genes having both expression and methylation values in AML as well as PC. Next, we run Jarque-Bera normality test on the expression/methylation data to divide the whole dataset into two parts: one that ollows normal distribution and the other that does not follow normal distribution. Thus, we have now four parts of the dataset: normally distributed expression data, normally distributed methylation data, non-normally distributed expression data, and non-normally distributed methylated data. A feature-extraction technique, "mRMR" is then utilized on each part. This results in a list of top-ranked genes. Next, we apply Welch t-test (parametric test) and Shrink t-test (non-parametric test) on the expression/methylation data for the top selected normally distributed genes and non-normally distributed genes, respectively. We then use a recent weighted ARM method, "RANWAR" to combine all/specific resultant genes to generate top oncogenic rules along with respective integrated markers. Finally, we perform literature search as well as KEGG pathway and Gene-Ontology (GO) analyses using Enrichr database for in silico validation of the prioritized oncogenes as the markers and labeling the markers as existing or novel.

UNASSIGNED

The novel markers of AML are {ABCB11↑∪KRT17↓} (i.e., ABCB11 as up-regulated, & KRT17 as down-regulated), and {AP1S1-∪KRT17↓∪NEIL2-∪DYDC1↓}) (i.e., AP1S1 and NEIL2 both as hypo-methylated, & KRT17 and DYDC1 both as down-regulated). The novel marker of PC is {UBIAD1¶∪APBA2‡∪C4orf31‡} (i.e., UBIAD1 as up-regulated and hypo-methylated, & APBA2 and C4orf31 both as down-regulated and hyper-methylated).

UNASSIGNED

The identified novel markers might have critical roles in AML as well as PC. The approach can be applied to other complex disease.

Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and often result in hepatic failure. There is no gold-standard test for diagnosing mitochondrial disease, and the current diagnosis relies on establishing a consistent pattern of evidence from clinical data, neuroimaging, tissue biopsy, and biochemical investigations. In some patients, the mitochondrial respiratory chain defect (MRCD) diagnosis is confirmed by genetic investigations. In most cases, genetic investigations are not informative and a number of cases remain unexplained.Here, we report on two children presenting with liver disease in whom first investigations suggested MRCD, due to decreased liver respiratory chain activities and decreased mitochondrial DNA copy number. However, sequencing of the genes known to be associated with mitochondrial DNA instability did not identify any pathogenic mutations. Further investigations including exome analysis, biliary bile salt analysis, and/or BSEP immunostaining detected a defect in the bile salt export pump (BSEP). Diagnosis of progressive familial intrahepatic cholestasis type 2 (PFIC2), a hereditary disorder in bile formation due to BSEP deficiency was confirmed by ABCB11 gene sequencing. Deleterious mutations were identified in both patients: one harboring compound heterozygous mutations (p.Arg470*/c.1308+2T>A) and the other homozygous nonsense mutation (p.Tyr354*). This report increases awareness of a possible secondary mitochondrial respiratory chain defect in the liver tissue associated with other underlying causes such as PFIC2.

OBJECTIVE

Although liver disease is a major complication of parenteral nutrition (PN) for intestinal failure (IF), its pathogenesis remains unclear. We investigated potential molecular mechanisms of liver injury in pediatric onset IF.

METHODS

Liver expression of canalicular phospholipid (ABCB4), bile acid (ABCB11), and sterol (ABCG5/8) transporters, their upstream regulators LXR and FXR as well as pro-inflammatory cytokines interleukin-6 (IL6) and tumor necrosis factor (TNF) were investigated among patients with IF [age median 3.8 (IQR 1.2 to 11)] in relation to biochemical and histologic liver injury, PN, serum plant sterols, fibroblast growth factor 19, and α-tocopherol.

RESULTS

Patients receiving PN currently (n = 18) showed more advanced liver injury than patients after weaning off PN (n = 30). Histologic portal inflammation strongly segregated PN-dependent (44%) from weaned off patients (3%, P = 0.001) and coupled with progression of cholestasis and liver fibrosis. Patients with portal inflammation demonstrated markedly induced liver RNA expression of IL6 and TNF, repression of FXR and its canalicular bile transporter target gene RNA expression, including ABCB4 and ABCB11 as well as decreased protein expression of ABCB11 and ABCB4. Furthermore, upregulation of LXR and ABCG5/8 RNA expression was suppressed in patients with portal inflammation. Current PN, increased serum levels of plant sterols stigmasterol, avenasterol, and sitosterol along with serum citrulline, a marker of enterocyte mass, predicted portal inflammation.

CONCLUSIONS

In pediatric onset IF, current PN delivery synergistically with intestinal compromise promote liver inflammation, which associates with progression of biochemical and histologic liver injury, while reducing expression of canalicular bile transporters.

OBJECTIVE

The bile salt export pump (BSEP, ABCB11) is essential for bile salt secretion at the canalicular membrane of liver cells. Clinical phenotypes associated with BSEP mutations are commonly categorized as benign recurrent intrahepatic cholestasis (BRIC-2) or progressive familial intrahepatic cholestasis (PFIC-2).

METHODS

The molecular basis of BSEP-associated liver disease in a sibling pair was characterized by immunostaining, gene sequencing, bile salt analysis and recombinant expression in mammalian cells and yeast for localization and in vitro activity studies respectively.

RESULTS

Benign recurrent intrahepatic cholestasis was considered in a brother and sister who both suffered from intermittent cholestasis since childhood. Gene sequencing of ABCB11 identified the novel missense mutation p.G374S, which is localized in the putative sixth transmembrane helix of BSEP. Liver fibrosis was present in the brother at the age of 18 with progression to cirrhosis within 3 years. Immunofluorescence of liver tissue showed clear canalicular BSEP expression; however, biliary concentration of bile salts was drastically reduced. In line with these in vivo findings, HEK293 cells showed regular membrane targeting of human BSEP(G374S), whereas in vitro transport measurements revealed a strongly reduced transport activity.

CONCLUSIONS

The novel mutation p.G374S impairs transport function without disabling membrane localization of BSEP. While all other known BSEP mutations within transmembrane helices are associated with PFIC-2, the new p.G374S mutation causes a transitional phenotype between BRIC-2 and PFIC-2.

We have reported previously that centrally applied ET (endothelin)-1 and ET-3 induce either choleresis or cholestasis depending on the dose. In the present study, we sought to establish the role of these endothelins in the short-term peripheral regulation of bile secretion in the rat. Intravenously infused endothelins induced significant choleresis in a dose-dependent fashion, ET-1 being more potent than ET-3. Endothelins (with the exception of a higher dose of ET-1) did not affect BP (blood pressure), portal venous pressure or portal blood flow. ET-1 and ET-3 augmented the biliary excretion of bile salts, glutathione and electrolytes, suggesting enhanced bile acid-dependent and -independent bile flows. ET-induced choleresis was mediated by ET(B) receptors coupled to NO and inhibited by truncal vagotomy, atropine administration and capsaicin perivagal application, supporting the participation of vagovagal reflexes. RT (reverse transcription)-PCR and Western blot analysis revealed ETA and ET(B) receptor expression in the vagus nerve. Endothelins, through ET(B) receptors, augmented the hepatocyte plasma membrane expression of Ntcp (Na⁺/taurocholate co-transporting polypeptide; Slc10a1), Bsep (bile-salt export pump; Abcb11), Mrp2 (multidrug resistance protein-2; Abcc2) and Aqp8 (aquaporin 8). Endothelins also increased the mRNAs of these transporters. ET-1 and ET-3 induced choleresis mediated by ET(B) receptors coupled to NO release and vagovagal reflexes without involving haemodynamic changes. Endothelin-induced choleresis seems to be caused by increased plasma membrane translocation and transcriptional expression of key bile transporters. These findings indicate that endothelins are able to elicit haemodynamic-independent biological effects in the liver and suggest that these peptides may play a beneficial role in pathophysiological situations where bile secretion is impaired.

Cell transplantation is a potential therapy for acquired or inherited liver diseases. Donor-derived hepatocytes (DDH) have been found in humans and mice after bone marrow transplantation (BMT) but with highly variable frequencies in different disease models. To test the effect of liver repopulation after BMT in inherited cholestatic liver diseases, spgp (sister of P-glycoprotein, or bile salt export pump, abcb11) knockout mice, a model for human progressive intrahepatic cholestasis type 2 with defects in excreting bile salts across the hepatocyte canalicular membrane, were transplanted with bone marrow cells from enhanced green fluorescent protein (EGFP) transgenic donor mice after lethal irradiation. One to 6 months later, scattered EGFP-positive DDHs with positive spgp staining were observed in the liver. These hepatocytes had been incorporated into hepatic plates and stained positively with hepatocyte-specific marker albumin. RT-PCR for the spgp gene revealed positive expression in the liver of sgsp knockout mice that had received the transplant. Bile acid analysis of bile samples showed that these mice also had higher levels of total biliary bile acid and taurocholic acid concentration than knockout mice without transplantation, indicating that BMT partially improved biliary bile acid secretion. Our results indicate that bone marrow cells could serve as a potential source for restoration of hepatic functions in chronic metabolic liver disease.

Hyperbilirubinemia is a frequent side effect induced by long-term therapy with the antibiotic fusidate. The aim of this study was to elucidate the molecular mechanisms of fusidate-induced hyperbilirubinemia by investigating its influence on hepatic transport systems in the canalicular membrane. Using canalicular membrane vesicles from rat liver, we determined the effect of fusidate on the adenosine 5'-triphosphate (ATP)-dependent transport of substrates of the apical conjugate export pump, multi-drug resistance protein 2 (Mrp2, symbol Abcc2) and the bile salt export pump (Bsep, symbol Abcb11). Fusidate inhibited the ATP-dependent transport of the Mrp2 substrates 17beta-glucuronosyl estradiol and leukotriene C4, and the transport of cholyltaurine by Bsep with Ki values of 2.2+/-0.3, 7.6+/-1.3, and 5.5+/-0.8 microM, respectively. To elucidate the in vivo implication of these findings, the effect of fusidate treatment on the elimination of intravenously administered tracer doses of 17beta-glucuronosyl estradiol and cholyltaurine into bile was studied in rats. Treatment with fusidate (100 micromol/kg body weight) reduced the biliary excretion rate of 17beta-glucuronosyl [3H]estradiol and [3H]cholyltaurine by 75 and 80%, respectively. Extended treatment of rats with fusidate (100 micromol/kg body weight, three times daily i.p. for 3 days) reduced hepatic Mrp2 protein levels by 61% (P<0.001). Our data suggest that there are at least two different mechanisms involved in the impairment of transport processes and hepatobiliary elimination by fusidate, direct inhibition of transport of Mrp2 and Bsep substrates by competitive interaction and impairment by a decreased level of hepatic Mrp2.

Despite considerable progress in identifying specific HLA alleles as genetic risk factors for some forms of drug-induced liver injury, progress in understanding whether genetic polymorphisms relevant to drug disposition also contribute to risk for developing this serious toxicity has been more limited. Evidence from both candidate-gene case control studies and genome-wide association studies is now discussed. In the case of genes relevant to drug metabolism, polymorphisms in cytochromes P450, UDP-glucuronosyltransferases, N-acetyltransferases and glutathione S-transferases as risk factors for DILI are assessed. The relevance of ABC transporters to drug-induced liver injury is also considered, together with data showing associations of particular ABCB11, ABCB1 and ABCC2 polymorphisms with some forms of drug-induced liver injury. Very few of the associations with genes relevant to drug disposition that have been reported have been well replicated. Even apparently well-studied associations such as that between isoniazid liver injury and N-acetyltransferase 2 slow acetylators remain problematic, though it seems likely that polymorphisms in drug metabolism genes do contribute to risk for some specific drugs. A better understanding of genetic risk factors for drug-induced liver injury will require further genome-wide association studies with larger numbers of cases, especially for forms of drug-induced liver injury where HLA genotype does not appear to be a risk factor.

Silibinin have been introduced for several years as a potent antioxidant in the field of nutraceuticals. Based on wide persuasive effects of this drug, we have decided to investigate the effects of silibinin on chronic myelogenous leukemia (CML) in vitro models, K562 and KCL22 cell lines. Lactate dehydrogenase (LDH) release, microculture tetrazolium test (MTT assay) and real-time PCR were employed to evaluate the effects of silibinin on cell cytotoxicity, cell proliferation and expression of various multidrug resistance genes in these cell lines, respectively. Our results have shown that presence of silibinin has inhibitory effects on cell proliferation of K562 and KCL22 cell lines. Also, our data indicated that silibinin, in a dose-dependent manner with applying no cytotoxic effects, inhibited cell proliferation and reduced mRNA expression levels of some transporter genes e.g. MDR1, MRP3, MRP2, MRP1, MRP5, MRP4, ABCG2, ABCB11, MRP6 and MRP7. The multifarious in vitro inhibitory effects of silibinin are in agreement with growing body of evidence that silibinin would be an efficient anticancer agent in order to be used in multi-target therapy to prevail the therapeutic hold backs against CML.

Modifiers of response to glucose, insulin and potassium (GIK) infusion may affect clinical outcomes in acute coronary syndromes (ACS). In an Immediate Myocardial Metabolic Enhancement During Initial Assessment And Treatment In Emergency Care (IMMEDIATE) trial's sub-study (n = 318), we explored effects of 132,634 genetic variants on plasma glucose and potassium response to 12-h GIK infusion. Associations between metabolite-associated variants and infarct size (n = 84) were assessed. The 'G' allele of rs12641551, near ACSL1, as well as the 'A' allele of XPO4 rs2585897 were associated with a differential glucose response (P for 2 degrees of freedom test, P2df ⩽ 4.75 × 10(-7)) and infarct size with GIK (P2df < 0.05). Variants within or near TAS1R3, LCA5, DNAH5, PTPRG, MAGI1, PTCSC3, STRADA, AKAP12, ARFGEF2, ADCYAP1, SETX, NDRG4 and ABCB11 modified glucose response, and near CSF1/AHCYL1 potassium response (P2df ⩽ 4.26 × 10(-7)), but not outcomes. Gene variants may modify glucose and potassium response to GIK infusion, contributing to cardiovascular outcomes in ACS.

OBJECTIVE

Single gene mutations cause syndromes of intrahepatic cholestasis, but previous multi-gene mutation screening in children with idiopathic cholestasis failed to fulfill diagnostic criteria in approximately two-thirds of children. In adults with fibrosing cholestatic disease, heterozygous ABCB4 mutations were present in 34% of patients. Here, we hypothesized that children with idiopathic cholestasis have a higher frequency of heterozygous non-synonymous gene sequence variants.

METHODS

We analyzed the frequency and types of variants in 717 children in whom high-throughput sequencing of the genes SERPINA1, JAG1, ATP8B1, ABCB11 and ABCB4 was performed as part of an evaluation for idiopathic intrahepatic cholestasis cholestasis. The frequency of non-synonymous variants (NSV) was compared with those of 1092 control subjects enrolled in the 1000 Genome Project.

RESULTS

The frequency of NSV in single genes was similar between disease (25%) and controls (26%, P = 0.518). In contrast, double or triple NSV in two or more genes were more frequent in disease (n = 7%) than controls (n = 4.7%, P = 0.028). Detailed review of clinical and laboratory information in a subgroup of double or triple heterozygous patients revealed variable γ-glutamyltransferase levels and severity of pruritus, with liver biopsies showing stage 2-3 fibrosis.

CONCLUSIONS

Children with idiopathic intrahepatic cholestasis have a higher frequency of double or triple NSV in SERPINA1, JAG1, ATPB1, ABCB11 or ABCB4. These findings raise the potential role for gene-gene relationships in determining the phenotype of cholestatic liver disease in children.

The present study sought to investigate the genetic variants in drug metabolizing enzyme and transporter (DMET) genes associated with steady-state plasma concentrations of risperidone among Thai autism spectrum disorder (ASD) patients. ASD patients taking risperidone for at least 1 month were enrolled for this pharmacogenomic study. Genotyping profile was obtained using Affymetrix DMET Plus array interrogating 1931 variants in 231 genes. Steady-state plasma risperidone and 9-hydroxyrisperidone were measured using liquid chromatography/tandem mass spectrometry assay. The final analysis included 483 markers for 167 genes. Six variants, ABCB11 (c.3084A>> G, c.∗420A>> G, c.∗368G>> A, and c.∗236G>> A) and ADH7 (c.690G>> A and c.-5360G>> A), were found to be associated with plasma concentrations of risperidone. 9-Hydroxyrisperidone and the total active-moiety levels were associated with six gene variants, SCLO1B1 (c.-11187G>> A and c.521T>> C), SLCO1B3 (c.334G>> T, c.699A>> G, and c.1557G>> A), and SLC7A5 c.∗438C>> G. Polymorphisms in UGT2B4 c.∗448A>> G and CYP2D6 (c.1661G>> C, c.4180G>> C, and c.-2178G>> A) showed considerable but not significant associations with metabolic ratio. This pharmacogenomic study identifies new genetic variants of DMET genes in monitoring risperidone therapy.