OATP1B1 Research Articles

English

Rifampin is one of first-line drugs for the treatment of tuberculosis. In Indonesia nearly all tuberculosis patients show lower rifampin plasma concentrations possibly due to genetics. Rifampin is a substrate of the organic anion-transporting polypeptide 1B1 (OATP 1B1) encoded by the solute carrier organic anion transporter family member 1B1 (SLCO1B1). This study aimed to identify haplotype polymorphisms of tuberculosis drug transporters with an impact on clinical outcome in tuberculosis patients. Thirty-six patients from Abdul Wahab Sjahranie General Hospital, Samarinda, East Kalimantan were involved in the study. Buffy coat from patient blood samples were tested for SLCO1B1 and SLCO1B3 polymorphisms by RFLP and ARMS PCR, whereas the clinical outcome was examined based on the sputum conversion. The frequency of patients with SLCO1B1*15 haplotype was 63.9%. The SLCO1B1*15 haplotype was associated with susceptibility to failure of clinical outcome (p=0.005; RR=4.52; 95% CI: 1.22-16.64). The OATP1B1*15 haplotype revealed that the failure of clinical outcome was markedly increased compared to the three other haplotypes. These results suggest that the SLCO1B1*15 haplotype is an important predisposing factor for lower clinical outcome. Our data indicate that individualized treatment should be considered for Indonesian tuberculosis patients based on genetics characteristics of patients.

Inhibition of intestinal ascorbic acid uptake by lipopolysaccharide is mediated via transcriptional mechanisms

Ascorbic acid (AA) accumulation in intestinal epithelial cells is an active transport process mainly mediated by two sodium-dependent vitamin C transporters (SVCT-1 and SVCT-2). To date, little is known about the effect of gut microbiota generated lipopolysaccharide (LPS) on intestinal absorption of water-soluble vitamins. Therefore, the objective of this study was to investigate the effects of bacterially-derived LPS on AA homeostasis in enterocytes using Caco-2 cells, mouse intestine and intestinal enteroids models. Pre-treating Caco-2 cells and mice with LPS led to a significant decrease in carrier-mediated AA uptake. This inhibition was associated with a significant reduction in SVCT-1 and SVCT-2 protein, mRNA, and hnRNA expression. Furthermore, pre-treating enteroids with LPS also led to a marked decrease in SVCT-1 and SVCT-2 protein and mRNA expression. Inhibition of SVCT-1 and SVCT-2 occurred at least in part at the transcriptional level as promoter activity of SLC23A1 and SLC23A2 was attenuated following LPS treatment. Subsequently, we examined the protein and mRNA expression levels of HNF1α and Sp1 transcription factors, which are needed for basal SLC23A1 and SLC23A2 promoter activity, and found that they were significantly decreased in the LPS treated Caco-2 cells and mouse jejunum; this was reflected on level of the observed reduction in the interaction of these transcription factors with their respective promoters in Caco-2 cells treated with LPS. Our findings indicate that LPS inhibits intestinal carrier- mediated AA uptake by down regulating the expression of both vitamin C transporters and transcriptional regulation of SLC23A1 and SLC23A2 genes.

Association between SLC19A1 Gene Polymorphism and High Dose Methotrexate Toxicity in Childhood Acute Lymphoblastic Leukaemia and Non Hodgkin Malignant Lymphoma: Introducing a Haplotype based Approach.

BackgroundWe investigated the clinical relevance of SLC 19A1 genetic variability for high dose methotrexate (HD-MTX) related toxicities in children and adolescents with acute lymphoblastic leukaemia (ALL) and non Hodgkin malignant lymphoma (NHML).Patients and methodsEighty-eight children and adolescents with ALL/NHML were investigated for the influence of SLC 19A1 single nucleotide polymorphisms (SNPs) and haplotypes on HD-MTX induced toxicities.ResultsPatients with rs2838958 TT genotype had higher probability for mucositis development as compared to carriers of at least one rs2838958 C allele (OR 0.226 (0.071–0.725), p < 0.009). Haplotype TGTTCCG (H4) statistically significantly reduced the risk for the occurrence of adverse events during treatment with HD-MTX (OR 0.143 (0.023–0.852), p = 0.030).ConclusionsSLC 19A1 SNP and haplotype analysis could provide additional information in a personalized HD-MTX therapy for children with ALL/NHML in order to achieve better treatment outcome. However further studies are needed to validate the results.

Vitamin K epoxide reductase complex subunit 1 (VKORC1): A pharmacogenomic predictor of response and survival in patients (pts) on triplet hepatic artery infusion (HAI) and intravenous cetuximab (IV-Cet) for initially unresectable liver metastases from colorectal cancer (uLM-CRC) (EU trial OPTILIV).

2569 Background: The HAI of Irinotecan-Oxaliplatin-5-Fluorouracil (IFO) with IV-Cet achieved 29.7% complete uLM-CRC resections (R0+R1) and an overall median survival (OS) of 25.7 months in previously treated pts (Lévi, Ann Oncol 2016). Methods: To identify pharmacogenomic predictors of outcomes, 207 single nucleotide polymorphisms (SNPs) from 34 pharmacology genes were analysed in blood mononuclear cells (ADME PGx, MassArray platform, Sequenom, USA). Relations between SNPs and tumor response, R0+R1, survival, and toxicities were tested using adjusted Mann Whitney, Fisher Exact, Log Rank tests and Hardy-Weinberg Equilibrium. Results: Pts (16F;36M; 33-76 yo; WHO performance status 0-1) received protocol treatment as 2nd (21 pts) or 3-4thline (31 pts). VKORC1 SNPs in promoter (rs9923231) and intron (rs9934438) were consistently associated with early and objective responses, and overall survival. For rs9923231, T/T (N = 8) as compared to C/T (N = 21) had greatest chance of achieving early response (50% vs 5%, p = 0.029) or 4-y survival (46% vs 0%, p = 0.006). VKORC1 SNPs also related to HA thrombosis (rs992331, T/T, 77% vs C/C, 30%, p = 0.04). In contrast, NAT2 SNPs (rs1041983 and rs1801280) were associated with up to 5-fold differences in R0-R1 resection rate. Statistically significant associations (p &lt; 0.05) of SNPs with clinical outcomes were found for oxydo-reduction (CYP2E1 and HA thrombosis; CYP2C9 and diarrhea; CYP2C19 and diarrhea, fatigue, and early response), conjugation (UGT1A1 and diarrhea; NAT2 and fatigue); and transport (ABCB1 or SLC0B3 and neutropenia; SLC22A1 and diarrhea; SLC 15A2 and early response). Conclusions: VKORC1 was highlighted for the first time, as a pharmacogenomic predictor of HAI efficacy for LM-CRC. Conversion-to-resection was associated with NAT2 polymorphism. VKORC1 γ-carboxylates vitamin K-dependent proteins. Its polymorphism guides personalized warfarin dosing. VKORC1 SNPs determination could help identify the uLM-CRC pts who best benefit from intensive HAI therapy. Clinical trial information: NCT00852228.

The SLC22A5 genetic analysis in Chinese patients with systemic primary carnitine deficiency

Objective To investigate the clinical and biochemical metabolic features of 12 patients with systemic primary carnitine deficiency(CDSP)and to identify the SLC22A5 gene mutation types of the disease. Method The clinical and biochemical data were collected by retrospective analysis. DNA direct sequencing and multiplex ligation dependent probe amplification(MLPA)were applied for SLC22A5 gene analysis. Result Among 12 patients with CDSP, 3 cases had evident infection factors, 6 cases with convulsions, 5 cases manifested liver hypertrophy, 8 cases with hyperammonemia, and 9 cases showed myocardial damage. All CDSP patients were detected biallelic pathogenic mutation in SLC22A5 gene by direct sequencing. The gene types include IVS2+ 1G>T, c. 3G>T(p.Met1Ile), c. 760C>T(p.Arg254X), c. 1400C>G(p.Ser467Cys), c. 844dupc(p.Arg282fs), c. 338G>A(p.Cys113Tyr), c. 51C>G(p.Phe17Leu), c. 659A>T(p.Glu220Val), and c. 1365dupC(p.Thr456fs). c. 659A>T(p.Glu220Val)and c. 1365dupC(p.Thr456fs)are novel mutations. One female patient was maternal CDSP, her child had abnormal newborn screening. The allele frequency of c. 760C>T(p.Arg254X)and c. 1400C>G(p.Ser467Cys)were 37.5%(9/24)and 29.2%(7/24)respectively. The MLPA test results of all patients were negative. Conclusion The clinical manifestations are complex and various in patients with CDSP. Point and small InDel(insertions/deletions)mutation constitute the major alteration in SLC22A5 gene. c. 1400C>G(p.Ser467Cys)might be another prevalence mutation type in Chinese CDSP patient. (Chin J Endocrinol Metab, 2017, 33: 208-214) Key words: Systemic primary carnitine deficiency; SLC22A5; Free carnitine; Acylcarnitine; Multiplex ligation dependent probe amplification

Extended Abstract: Deficiency of Sodium Taurocholate Cotransporting Polypeptide (SLC10A1): A New Inborn Error of Metabolism with an Attenuated Phenotype

We present the first patient with a defect in the Na⁺-taurocholate cotransporting polypeptide SLC10A1 (NTCP), which plays a key role in the enterohepatic circulation of bile salts. The clinical presentation of the child was mild and the child showed no signs of liver dysfunction or pruritus despite extremely elevated plasma bile salt levels (>100-fold upper-limit of normal). A homozygous point mutation was found in the SLC10A1 gene (resulting in amino acid change R252H) and functional studies confirmed the pathogenicity of the mutation. This confirms the role of NTCP as the major transporter of conjugated bile salts into the liver as part of the enterohepatic circulation and shows that other transporters partly can take over its function, resulting in a relatively mild phenotype. This work was published previously in [Vaz et al.: Hepatology 2015;61:260-267] and supplemented with some follow-up information of the patient.

Na+-taurocholate cotransporting polypeptide (NTCP/SLC10A1) ortholog in the marine skate Leucoraja erinacea is not a physiological bile salt transporter.

The Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1) is a hepatocyte-specific solute carrier, which plays an important role in maintaining bile salt homeostasis in mammals. The absence of a hepatic Na+-dependent bile salt transport system in marine skate and rainbow trout raises a question regarding the function of the Slc10a1 gene in these species. Here, we have characterized the Slc10a1 gene in the marine skate, Leucoraja erinacea The transcript of skate Slc10a1 (skSlc10a1) encodes 319 amino acids and shares 46% identity to human NTCP (hNTCP) with similar topology to mammalian NTCP. SkSlc10a1 mRNA was mostly confined to the brain and testes with minimal expression in the liver. An FXR-bile salt reporter assay indicated that skSlc10a1 transported taurocholic acid (TCA) and scymnol sulfate, but not as effectively as hNTCP. An [3H]TCA uptake assay revealed that skSlc10a1 functioned as a Na+-dependent transporter, but with low affinity for TCA (Km = 92.4 µM) and scymnol sulfate (Ki = 31 µM), compared with hNTCP (TCA, Km = 5.4 µM; Scymnol sulfate, Ki = 3.5 µM). In contrast, the bile salt concentration in skate plasma was 2 µM, similar to levels seen in mammals. Interestingly, skSlc10a1 demonstrated transport activity for the neurosteroids dehydroepiandrosterone sulfate and estrone-3-sulfate at physiological concentration, similar to hNTCP. Together, our findings indicate that skSlc10a1 is not a physiological bile salt transporter, providing a molecular explanation for the absence of a hepatic Na+-dependent bile salt uptake system in skate. We speculate that Slc10a1 is a neurosteroid transporter in skate that gained its substrate specificity for bile salts later in vertebrate evolution.

Molecular mechanism(s) involved in differential expression of vitamin C transporters along the intestinal tract.

Mammalian cells utilize two transporters for the uptake of ascorbic acid (AA), Na+-dependent vitamin C transporter SVCT-1 and SVCT-2. In the intestine, these transporters are involved in AA absorption and are expressed at the apical and basolateral membrane domains of the polarized epithelia, respectively. Little is known about the differential expression of these two transporters along the anterior-posterior axis of the intestinal tract and the molecular mechanism(s) that dictate this pattern of expression. We used mouse and human intestinal cDNAs to address these issues. The results showed a significantly lower rate of carrier-mediated AA uptake by mouse colon than jejunum. This was associated with a significantly lower level of expression of SVCT-1 and SVCT-2 at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels in the colon than the jejunum, implying the involvement of transcriptional mechanism(s). Similarly, expression levels of SVCT-1 and SVCT-2 mRNA and hnRNA were significantly lower in human colon. We also examined the levels of expression of hepatocyte nuclear factor 1α and specificity protein 1, which drive transcription of the Slc23a1 and Slc23a2 promoters, respectively, and found them to be markedly lower in the colon. Furthermore, significantly lower levels of the activating markers for histone (H3) modifications [H3 trimethylation of lysine 4 (H3K4me3) and H3 triacetylation of lysine 9 (H3K9ac)] were observed in the Slc23a1 and Slc23a2 promoters in the colon. These findings show, for the first time, that SVCT-1 and SVCT-2 are differentially expressed along the intestinal tract and that this pattern of expression is, at least in part, mediated via transcriptional/epigenetic mechanisms.NEW & NOTEWORTHY Our findings show, for the first time, that transporters of the water-soluble vitamin ascorbic acid (i.e., the vitamin C transporters SVCT-1 and SVCT-2) are differentially expressed along the length of the intestinal tract and that the pattern of expression is mediated, at least in part, by transcriptional and epigenetic mechanism(s) affecting both Slc23a1 and Slc23a2 genes.

Clinical and molecular study of a pediatric patient with sodium taurocholate cotransporting polypeptide deficiency.

The human solute carrier family 10 member 1 (SLC10A1) gene encodes sodium taurocholate cotransporting polypeptide (NTCP), the principal transporter of conjugated bile salts from the plasma into hepatocytes. Although the function of NTCP has been studied extensively and a number of SLC10A1 variations have been identified in humans, information regarding NTCP deficiency is limited. To date, only one patient with NTCP deficiency has been described; however, in the present study a pediatric patient who experienced intractable and striking hypercholanemia is presented. Analysis of the SLC10A1 gene in the patient revealed a homozygous p.Ser267Phe (c.800C>T) variation, which proved to be a single-nucleotide polymorphism (SNP) in the allele frequency of 4.7% of healthy controls. This variation involved a conserved amino acid residue on the orthologous alignment that was predicted to be ‘disease-causing’ by functional analysis using a number of bioinformatic tools. Next generation sequencing was performed; however, no other genetic causes were identified that would affect the bile acid homeostasis in the patient. Moreover, an adult, with the same genotype as the pediatric patient, was identified for the first time as experiencing mild hypercholanemia. The molecular and clinical findings in the present study suggest, for the first time, that there is an association between p.Ser267Phe SNP and hypercholanemia, and this information may be used to clinically identify NTCP deficiency worldwide.

A study of correlation between uric acid transporter gene SLC16A9 polymorphism and hyperuricemia in Uyghur

Objective The transport and excretion of uric acid play significant role in occurrence of hyperuricemia. The correlation between polymorphism of the gene(SLC16A9)and the occurrence of hyperuricemia was explored in this study. Methods Case-control study was used in this research in which 1 026 patients with hyperuricemia were collected in the hospital, while 1 031 control cases were selected from regular medical check-ups. Sequenom MassARRAY® SNP was utilized in both hyperuricemia and control groups to detect the correlation between 4 SNP sites of SLC16A9 mutation related to uric acid excretion in order to analyze the association between gene mutation and hyperuricemia among Uyghur. Results In males, blood uric acid has a positive association with body mass index, waist circumferences, fasting blood glucose(r=0.140, 0.237, 0.084, all P 0.05). As compared with GG genotype, AG heterozygote has a high risk(OR=1.991, 95%CI 1.032-3.842, P<0.01). More importantly, rs4948351 stie revealed that TT and CT also had a higher risk(OR=1.453, 95%CI 1.079-1.957; OR=1.353, 95%CI 1.007-1.818), and there was a significant difference between two groups(P=0.034), suggesting that rs1171614 and rs4948351 associated with hyperuricemia. Conclusion The study further confirms the relationship between the SLC16A9 gene and the pathogenesis of hyperuricemia. (Chin J Endocrinol Metab, 2016, 32: 743-748) Key words: Hyperuricemia; Gene polymorphism; SLC16A9; Uric acid transporter gene; Uygur

Clarification of the Mechanism of Clopidogrel-Mediated Drug-Drug Interaction in a Clinical Cassette Small-dose Study and Its Prediction Based on In Vitro Information.

Clopidogrel is reported to be associated with cerivastatin-induced rhabdomyolysis, and clopidogrel and its metabolites are capable of inhibiting CYP2C8 and OATP 1B1 in vitro. The objective of the present study was to identify the mechanism of clopidogrel-mediated drug-drug interactions (DDIs) on the pharmacokinetics of OATP1B1 and/or CYP2C8 substrates in vivo. A clinical cassette small-dose study using OATPs, CYP2C8, and OATP1B1/CYP2C8 probe drugs (pitavastatin, pioglitazone, and repaglinide, respectively) with or without the coadministration of either 600 mg rifampicin (an inhibitor for OATPs), 200 mg trimethoprim (an inhibitor for CYP2C8), or 300 mg clopidogrel was performed, and the area under the concentration-time curve (AUC) ratios (AUCRs) for probe substrates were predicted using a static model. Clopidogrel increased the AUC of pioglitazone (2.0-fold) and repaglinide (3.1-fold) but did not significantly change the AUC of pitavastatin (1.1-fold). In addition, the AUC of pioglitazone M4, a CYP2C8-mediated metabolite of pioglitazone, was reduced to 70% of the control by coadministration of clopidogrel. The predicted AUCRs using the mechanism-based inhibition of CYP2C8 by clopidogrel acyl-β-glucuronide were similar to the observed AUCRs, and the predicted AUCR (1.1) of repaglinide using only the inhibition of OATP1B1 did not reach the observed AUCR (3.1). In conclusion, a single 300 mg of clopidogrel mainly inhibits CYP2C8-mediated metabolism by clopidogrel acyl-β-glucuronide, but its effect on the pharmacokinetics of OATP1B1 substrates is negligible. Clopidogrel is expected to have an effect not only on CYP2C8 substrates, but also dual CYP2C8/OATP1B1 substrates as seen in the case of repaglinide.

MicroRNA-Based Linkage between Aging and Cancer: from Epigenetics View Point.

Ageing is a complex process and a broad spectrum of physical, psychological, and social changes over time. Accompanying diseases and disabilities, which can interfere with cancer treatment and recovery, occur in old ages. MicroRNAs (miRNAs) are a set of small non-coding RNAs, which have considerable roles in post-transcriptional regulation at gene expression level. In this review, we attempted to summarize the current knowledge of miRNAs functions in ageing, with mainly focuses on malignancies and all underlying genetic, molecular and epigenetics mechanisms. The evidences indicated the complex and dynamic nature of miRNA-based linkage of ageing and cancer at genomics and epigenomics levels which might be generally crucial for understanding the mechanisms of age-related cancer and ageing. Recently in the field of cancer and ageing, scientists claimed that uric acid can be used to regulate reactive oxygen species (ROS), leading to cancer and ageing prevention; these findings highlight the role of miRNA-based inhibition of the SLC2A9 antioxidant pathway in cancer, as a novel way to kill malignant cells, while a patient is fighting with cancer.

Uptake of ascorbic acid by pancreatic acinar cells is negatively impacted by chronic alcohol exposure.

Vitamin C (ascorbic acid, AA) is indispensable for normal metabolism of all mammalian cells including pancreatic acinar cells (PACs). PACs obtain AA from their surroundings via transport across the cell membrane. Chronic alcohol exposure negatively affects body AA homeostasis; it also inhibits uptake of other micronutrients into PACs, but its effect on AA uptake is not clear. We examined this issue using both in vitro (266-6 cells) and in vivo (mice) models of chronic alcohol exposure. First, we determined the relative expression of the AA transporters 1 and 2 [i.e., sodium-dependent vitamin C transporter-1 (SVCT-1) and SVCT-2] in mouse and human PACs and found SVCT-2 to be the predominant transporter. Chronic exposure of 266-6 cells to alcohol significantly inhibited AA uptake and caused a marked reduction in SVCT-2 expression at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Similarly, chronic alcohol feeding of mice significantly inhibited AA uptake and caused a marked reduction in level of expression of the SVCT-2 protein, mRNA, and hnRNA. These findings suggest possible involvement of transcriptional mechanism(s) in mediating chronic alcohol effect on AA uptake by PACs. We also observed significant epigenetic changes (histone modifications) in the Slc23a2 gene (reduction in H3K4me3 level and an increase in H3K27me3 level) in the alcohol-exposed 266-6 cells. These findings show that chronic alcohol exposure inhibits PAC AA uptake and that the effect is mediated, in part, at the level of transcription of the Slc23a2 gene and may involve epigenetic mechanism(s).

Effect and mechanism of Cyclosporin A and cholic acid on reducing the amanitin-induced human liver cell damage

Objective To explore the effect and mechanism of Cyclosporin A(CsA) and cholic acid on redu-cing the human liver cell damage induced by α-amanitin(AMA). Methods According to the previous research results, the minimum hepatocellular survival concentration against α-AMA (1.4 g/L), the experiment was conducted in 5 groups: control group, damage group, glycochenodeoxycholic acid group, CsA group, and CsA combined with cholic acid group (CsA+ taurocholic acid, CsA+ chenodeoxycholic acid, CsA+ glycocholic acid, CsA+ glycochenodeoxycholic acid, and CsA+ taurochenodeoxycholic acid). For each group, there were 3 time points for observation (24 h, 48 h and 72 h after attacking), CsA and CsA+ glycochenodeoxycholic acid was used to protect hepatocytes, respectively, and morphological changes in cells were observed with inverted phase contrast microscope, and the live cells were counted by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide(MTT)method, and aspertate aminotransferase (AST) and alanine aminotransferase (ALT) in the culture supernatant were detected by biochemical method. Results Compared with the control group, hepatocellular growth in the injury group was obviously suppressed, with progressive cellular apoptosis and significantly decreased absorbance value of MTT(0.345±0.021); the activity of AST and ALT increased gradually, reaching the highest after 72 h [(98.4±6.7) U/L and (116.2±9.5) U/L, respectively]. Compared with injury group, broken organelles decreased significantly and absorbance value elevated in glycochenodeoxycholic acid group, CsA group and CsA combined with cholic acid group, and at each time point, the highest absorbance value in the CsA+ taurochenodeoxycholic acid group[the highest was (0.656±0.014), P<0.05]; at the same time, the activity of AST and ALT didn't increase obviously, at each time point, the lowest in CsA+ taurochenodeoxycholic acid group[the lowest was (22.3±6.2) U/L and (20.2±5.4) U/L, P<0.05, respectively]. Conclusions CsA, as well as cholic acid, can protect human liver cells from the attack of α-AMA.The mechanism may be CsA, as an organic anion transfer peptide in humans (OATP1B1 and OATP1B3) suppressant, inhibits the absorption of α-AMA.The joint application of CsA and taurochenodeoxycholic acid is superior to the single OATP substrate or inhibitor. Key words: Cyclosporine A; Cholic acid; Human liver cell; Amanitin; Damage

Effect of Chronic Alcohol Exposure on Physiological and Molecular Aspects of Pancreatic Acinar Cells Ascorbic Acid Transport

Vitamin C (ascorbic acid, AA) is required for normal cellular metabolism including that of pancreatic acinar cells (PAC). The vitamin is a potent antioxidant and acts as a cofactor for several important enzymes, while low intracellular level of AA leads to oxidative stress. PAC obtain the vitamin from their surroundings via transport across their cell membrane. A negative effect of chronic alcohol exposure on uptake of AA that is important for reducing cellular oxidative stress may contribute to the known deleterious effects of alcohol on the pancreas. Recent studies have shown that such alcohol exposure affects PAC uptake of other micronutrients, however its effect on PAC AA uptake is not clear. We address this issue in the present study using mouse‐derived pancreatic acinar 266‐6 cells (in vitro exposure; 50 mM ethanol for 96hrs) and mice fed alcohol chronically and controls were pair‐fed the same diet without alcohol (in vivo exposure; 25%, v/v for 4 weeks). We first determined the relative expression of the two known AA transporters in mouse and human PAC, the sodium‐dependent vitamin C transporter‐1 and ‐2 (SVCT‐1 and SVCT‐2; products of the SLC23A1 and SLC23A2 genes, respectively) and found SVCT‐2 to be the predominant transporter in PAC. Chronic exposure of 266‐6 cells to alcohol significantly inhibited AA uptake, and this inhibition was associated with a marked reduction in SVCT‐2 expression at the protein, mRNA, and heterogenous nuclear RNA (hnRNA) levels. Also, chronic alcohol feeding of mice led to a significant reduction in the level of expression of SVCT‐2 protein, mRNA and hnRNA in PAC. These findings suggest the possible involvement of transcriptional mechanism(s) in mediating the chronic alcohol effect and this observation was supported by the significant epigenetic changes (histone modifications) affecting the Slc23a2 promoter (a decrease in euchromatin H3K4me3 and an increase in heterochromatin H3K27me3) that we found in the alcohol exposed 266‐6 cells compared to controls. No significant changes in the methylation status of the predicted CpG island within the Slc23a2 promoter was found. Collectively, these findings show that chronic alcohol exposure inhibits AA uptake by PAC and that the effect is in part mediated at the level of transcription of the Slc23a2 gene and involves epigenetic mechanism(s).Support or Funding InformationSupported by grants from the DVA and the NIH (AA18071, DK56061, and DK58057).

Endotoxin-Mediated Downregulation of Hepatic Drug Transporters in HIV-1 Transgenic Rats.

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

Genome wide association study of uric acid in Indian population and interaction of identified variants with Type 2 diabetes.

Abnormal level of Serum Uric Acid (SUA) is an important marker and risk factor for complex diseases including Type 2 Diabetes. Since genetic determinant of uric acid in Indians is totally unexplored, we tried to identify common variants associated with SUA in Indians using Genome Wide Association Study (GWAS). Association of five known variants in SLC2A9 and SLC22A11 genes with SUA level in 4,834 normoglycemics (1,109 in discovery and 3,725 in validation phase) was revealed with different effect size in Indians compared to other major ethnic population of the world. Combined analysis of 1,077 T2DM subjects (772 in discovery and 305 in validation phase) and normoglycemics revealed additional GWAS signal in ABCG2 gene. Differences in effect sizes of ABCG2 and SLC2A9 gene variants were observed between normoglycemics and T2DM patients. We identified two novel variants near long non-coding RNA genes AL356739.1 and AC064865.1 with nearly genome wide significance level. Meta-analysis and in silico replication in 11,745 individuals from AUSTWIN consortium improved association for rs12206002 in AL356739.1 gene to sub-genome wide association level. Our results extends association of SLC2A9, SLC22A11 and ABCG2 genes with SUA level in Indians and enrich the assemblages of evidence for SUA level and T2DM interrelationship.

High salt-diet reduces SLC14A1 gene expression in the choroid plexus of Dahl salt sensitive rats

Elevated Na+ concentration ([Na+]) in the cerebrospinal fluid (CSF) contributes to the development of salt-sensitive hypertension. CSF is formed by the choroid plexus (CP) in cerebral ventricles, and [Na+] in CSF is controlled by transporters in CP. Here, we examined the effect of high salt diet on the expression of urea transporters (UTs) in the CP of Dahl S vs Dahl R rats using real time PCR. High salt intake (8%, for 2 weeks) did not alter the mRNA levels of UT-A (encoded by SLC14A2 gene) in the CP of either Dahl S or Dahl R rats. In contrast, the mRNA levels of UT-B (encoded by SLC14A1 gene) were significantly reduced in the CP of Dahl S rats on high salt diet as compared with Dahl R rats or Dahl S rats on normal salt diet. Reduced UT-B expression was associated with increased [Na+] in the CSF and elevated mean arterial pressure (MAP) in Dahl S rats treated with high salt diet, as measured by radiotelemetry. High salt diet-induced reduction in UT-B protein expression in the CP of Dahl S rats was confirmed by Western blot. Immunohistochemistry using UT-B specific antibodies demonstrated that UT-B protein was expressed on the epithelial cells in the CP. These data indicate that high salt diet induces elevations in CSF [Na+] and in MAP, both of which are associated with reduced UT-B expression in the CP of Dahl S rats, as compared with Dahl R rats. The results suggest that altered UT-B expression in the CP may contribute to an imbalance of water and electrolytes in the CSF of Dahl S rats on high salt diet, thereby leading to alterations in MAP.

Molecular analysis of a single nucleotide polymorphism in SCL17A1 and uric acid levels in Uyghur patients with hyperuricemia in Xinjiang region

The association between the polymorphism of SLC17A1 gene and hyperuricemia in Uyghur population in Xinjiang region was explored by Chi-square test. A total of 1 024 patients with hyperuricemia and 1 033 healthy volunteers were included.The genotype frequencies of CC, CT, and TT in hyperuricemia and healthy controls were 24.9%, 53.14%, 21.96% and 29.7%, 47.52%, 22.77%, respectively. There was statistical difference in SLC17A1 rs9467596 genotype frequencies between hyperuricemia cases and controls(χ2=7.492, P=0.024). CT genotype could increase the risk of hyperuricemia compared with CC genotype(OR=1.334, 95%CI 1.082-1.644). No statistical significance among the genotypes was found in age, body mass index, blood pressure, blood glucose, triglyceride, total cholesterol, High-density lipoprotein-cholesterol, low-density lipoprotein-Cholesterd, blood urea nitrogen, and Creatinine. The polymorphism of rs9467596 in SLC17A1 may be a genetic marker to assess risk of hyperuricemia among Xinjiang Uyghur population. (Chin J Endocrinol Metab, 2015, 31: 254-256) Key words: Uyghur; Hyperuricemia; SLC17A1; Polymorphism, single nucleotide

Interaction of human organic anion transporter 2 (OAT2) and sodium taurocholate cotransporting polypeptide (NTCP) with antineoplastic drugs

The ability of an antineoplastic drug to exert its cytostatic effect depends largely on the balance between its uptake into and extrusion from the cancer cells. ATP driven efflux transporter proteins drive the export of antineoplastic drugs and play a pivotal role in the development of chemoresistance. As regards uptake transporters, comparably less is known on their impact in drug action. In the current study, we characterized the interactions of two uptake transporter proteins, expressed mainly in the liver; the organic anion transporter 2 (OAT2, encoded by the SLC22A7 gene) and the sodium taurocholate cotransporting polypeptide (NTCP, encoded by the SLC10A1 gene), stably transfected in human embryonic kidney cells, with some antineoplastic agents that are routinely being used in cancer chemotherapy. Whereas NTCP did not show any strong interactions with the cytostatics tested, we observed a very strong inhibition of OAT2 mediated [3H] cGMP uptake in the presence of bendamustine, irinotecan and paclitaxel. The Ki values of OAT2 for bendamustine, irinotecan and paclitaxel were determined to be 43.3±4.33μM, 26.4±2.34μM and 10.4±0.45μM, respectively. Incubation of bendamustine with OAT2 expressing cells increased the caspase-3 activity, and this increase was inhibited by simultaneous incubation with bendamustine and probenecid, a well-known inhibitor of OATs, suggesting that bendamustine is a substrate of OAT2. A higher accumulation of irinotecan was observed in OAT2 expressing cells compared to control pcDNA cells by HPLC analysis of cell lysates. The accumulation was diminished in the presence of cGMP, the substrate we used to functionally characterize OAT2, suggesting specificity of this uptake and the fact that OAT2 mediates uptake of irinotecan.