27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.

Galectin-9 Promotes Neuronal Restoration via Binding TLR-4 in a Rat Intracerebral Hemorrhage Model.

Intracerebral hemorrhage (ICH) is a devastating disease with high rates of mortality and morbidity. Galactose lectin-9 (Gal-9) belongs to the family of ß-galactoside-binding lectins, which has been shown to play a vital role in immune tolerance and inflammation. However, the function of Gal-9 in ICH has not been fully studied in details. Several experiments were carried out to explore the role of Gal-9 in the late period of ICH. Primarily, ICH models were established in male adult Sprague Dawley (SD) rats. Next, the relative protein levels of Gal-9 at different time points after ICH were examined and the result showed that the level of Gal-9 increased and peaked at the 7th day after ICH. Then we found that when the content of Gal-9 increased, both the number of M2-type microglia and the corresponding anti-inflammatory factors also increased. Through co-immunoprecipitation (CO-IP) analysis, it was found that Gal-9 combines with Toll-like receptor-4 (TLR-4) during the period of the recovery after ICH. TUNEL staining and Fluoro-Jade B staining (FJB) proved that the amount of cell death decreased with the increase of Gal-9 content. Additionally, several behavioral experiments also demonstrated that when the level of Gal-9 increased, the motor, sensory, learning, and memory abilities of the rats recovered better compared to the ICH group. In short, this study illustrated that Gal-9 takes a crucial role after ICH. Enhancing Gal-9 could alleviate brain injury and promote the recovery of ICH-induced injury, so that Gal-9 may exploit a new pathway for clinical treatment of ICH.

Expression of nuclear factor erythroid 2-related factor 2 following traumatic brain injury in the human brain.

Secondary brain injury imposes great effects on the prognosis of patients following traumatic brain injury (TBI). Accumulating evidence suggests that nuclear factor erythroid 2-related factor 2 (Nrf2) could play a neuroprotective role in experimental TBI models by regulating the expression of numerous antioxidant, anti-inflammatory, and neuroprotective proteins. However, whether Nrf2 is activated in patients following TBI is still unknown. In this study, human brain tissues were obtained during surgery from patients suffering from TBI. The purpose of this study was to investigate the expression of Nrf2 and Nrf2-regulated gene products, NAD(P)H quinine oxidoreductase 1, and glutathione S-transferase in human injured brain tissue after TBI. Our results revealed that the nuclear level of Nrf2 was significantly increased in injured brain tissues, whereas the cytoplasmic level of Nrf2 was markedly decreased. In addition, the expression of NAD(P)H quinine oxidoreductase 1 and glutathione S-transferase was significantly upregulated. Nrf2 may be activated and confer neuroprotection against secondary brain injury following TBI. Therefore, Nrf2 could serve as a promising molecular target for the treatment of TBI.

Protein expression and function of organic anion transporters in short-term and long-term cultures of Huh7 human hepatoma cells.

Human-derived hepatic cell lines are a valuable alternative to primary hepatocytes for drug metabolism, transport and toxicity studies. However, their relevance for investigations of drug-drug and drug-organic anion (e.g., bile acid, steroid hormone) interactions at the transporter level remains to be established. The aim of the present study was to determine the suitability of the Huh7 cell line for transporter-dependent experiments. Huh7 cells were cultured for 1 to 4 weeks and subsequently were analyzed for protein expression, localization and activity of solute carrier (SLC) and ATP-binding cassette (ABC) transporters involved in organic anion transport using liquid chromatography-tandem mass spectroscopy, immunocytochemistry, and model substrates [3H]taurocholate (TCA), [3H]dehydroepiandrosterone sulfate (DHEAS) and 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) diacetate. The extended 4-week culture resulted in a phenotype resembling primary hepatocytes and differentiated HepaRG cells: cuboidal hepatocyte-like cells with elongated bile canaliculi-like structures were surrounded by epithelium-like cells. Protein expression of OSTα, OSTß and OATP1B3 increased over time. Moreover, the uptake of the SLC probe substrate DHEAS was higher in 4-week than in 1-week Huh7 cultures. NTCP, OATP1B1, BSEP and MRP3 were barely or not detectable in Huh7 cells. OATP2B1, MRP2 and MRP4 protein expression remained at similar levels over the four weeks of culture. The activity of MRP2 and the formation of bile canaliculi-like structures were confirmed by accumulation of CDF in the intercellular compartments. Results indicate that along with morphological maturation, transporters responsible for alternative bile acid secretion pathways are expressed and active in long-term cultures of Huh7 cells, suggesting that differentiated Huh7 cells may be suitable for studying the function and regulation of these organic anion transporters.

Influence of Orally Administered Borneol on the Expression of Hepatic Transporters in Rats.

BACKGROUND AND OBJECTIVE: Borneol, a traditional Chinese medicine (TCM), is often orally co-administered with other TCM and chemical drugs, but the drug-drug interactions between borneol and the other compounds remains unclear. This work investigates the effect of orally administered borneol on the transcription and expression of hepatic uptake transporters (Ntcp, Oatp2b1, Oatp1a1, Oatp1a4, Oct1, Oct2, Octn2 and Oat2) and efflux transporters (Mdrla, Mrp2, Mrp4 and Mrp5) in rats, aiming to obtain essential information to guide its clinical applications. METHODS: Rats were administered borneol (33, 100 and 300 mg/kg/day, respectively) and vehicle (control) orally via intragastric gavage for 7 consecutive days. The mRNA levels of rat hepatic uptake transporters (Ntcp, Oatp2b1, Oatp1a1, Oatp1a4, Oct1, Oct2, Octn2 and Oat2) and efflux transporters (Mdrla, Mrp2, Mrp4 and Mrp5) were determined using real-time quantitative PCR, while the hepatic Ntcp, Mdrla, Mrp2, Mrp4 and Mrp5 proteins were quantified using western blotting. RESULTS: The oral administration of borneol led to dose-dependent inhibition of mRNA and protein expression of Mrp4 and Mdr1a, dose-independent inhibition of mRNA and protein expression of Mrp2, and inverse dose-dependent inhibition of mRNA and protein expression of Ntcp. No significant effects were observed for mRNA expression of the other transporters tested following borneol administration. CONCLUSIONS: Oral administration of borneol may affect the metabolism of substances that are involved in bile acid enterohepatic circulation and substrates of Ntcp, Mdrla, Mrp2 and Mrp4 transporters.

Solute carrier organic anion transporter family member 4A1 (SLCO4A1) as a prognosis marker of colorectal cancer.

PURPOSE: Solute carrier organic anion transporter family member 4A1 (SLCO4A1) is involved in the transport of various compounds, including sugars, bile salts, organic acids, metal ions, amine compounds, and estrogen. SLCO4A1 is highly expressed in several cancers and a gender bias has been observed in colorectal cancer (CRC). We investigated SLCO4A1 expression, its prognostic value in patients with CRC, and its role in CRC cell proliferation and metastasis. METHODS: SLCO4A1 expression was assessed by immunohistochemistry (IHC) on specimens from 84 patients with CRC. The association of SLCO4A1 expression with clinicopathological features was examined. To confirm the biological role of SLCO4A1 in CRC, four CRC cell lines expressing SLCO4A1 were used and SLCO4A1 expression was knocked down by siRNA. Cell proliferation, MTT, migration, invasion, and semisolid agar colony formation assays were performed. RESULTS: SLCO4A1 was overexpressed in 32% of the CRC samples. SLCO4A1 overexpression and pathologic T stage were independent prognostic factors of decreased survival (P = 0.021). Kaplan-Meier analysis indicated a decreased cumulative survival for patients highly expressing SLCO4A1 compared to patients showing low SLCO4A1 expression (Log-rank test, P = 0.025). In cell lines, SLCO4A1 knockdown resulted in a significant decrease of viability, invasion, and migration when compared to control cells. Semisolid colony formation assay indicated that SLCO4A1-knocked down cells presented poor carcinogenic abilities compared to control cells. CONCLUSIONS: SLCO4A1 may be a valuable marker of poor prognostic for CRC. Furthermore, SLCO4A1 plays an important role in CRC cell proliferation, migration, invasion, and carcinogenesis.

Construction of HEK293 cells stably expressing wild-type organic anion transporting polypeptide 1B1 (OATP1B1*1a) and variant OATP1B1*1b and OATP1B1*15.

A transgenic cell line stably expressing the human organic anion transporting polypeptide (OATP1B1) was established. Human Embryonic Kidney 293 (HEK293) cell line stably expressing OATP1B1*1a sequence was amplified through PCR with the extracted total RNA as templates from human liver, then subcloned into the plasmid pMD19-T and verified by sequencing. OATP1B1*1b/OATP1B1*15 mutant sequences were obtained by site-directed mutation PCR with pMD19-T/ OATP1B1*1a as templates. The plasmids pcDNA3.1(+)/OATP1B1*1a, *1b and *15 were constructed and transfected into HEK293 cell line using Lipofectamine 2000 transfection reagent. Several stable transfected clones were obtained after selection with G418. Using rosuvastatin as a probe substrate of OATP1B1, the intracellular rosuvastatin accumulation in HEK293 and HEK-OATP1B1*1a, *1b and *15 monoclone cells were validated by a ultra-performance liquid chromatography-tandem mass spectrometry. OATP1B1 mRNA and protein expression were detected by RT-PCR and Western blot, respectively. The results from RT-PCR, rosuvastatin uptake and Western blot assay indicated that human OATP1B1 was highly expressed in transfected cells compared with controls. The HEK-293 cell lines stably expressing human OATP1B1-wild and variant (HEK-OATP1B1, *1b and *15) are potential models to study drug transport in vitro.

Comparison of mercury sulfides with mercury chloride and methylmercury on hepatic P450, phase-2 and transporter gene expression in mice.

Zuotai (mainly ß-HgS) and Zhusha (also called as cinnabar, mainly α-HgS) are used in traditional medicines in combination with herbs or even drugs in the treatment of various disorders, while mercury chloride (HgCl2) and methylmercury (MeHg) do not have known medical values but are highly toxic. This study aimed to compare the effects of mercury sulfides with HgCl2 and MeHg on hepatic drug processing gene expression. Mice were orally administrated with Zuotai (ß-HgS, 30mg/kg), α-HgS (HgS, 30mg/kg), HgCl2 (33.6mg/kg), or MeHg (3.1mg/kg) for 7days, and the expression of genes related to phase-1 drug metabolism (P450), phase-2 conjugation, and phase-3 (transporters) genes were examined. The mercurials at the dose and duration used in the study did not have significant effects on the expression of cytochrome P450 1-4 family genes and the corresponding nuclear receptors, except for a slight increase in PPARα and Cyp4a10 by HgCl2. The expressions of UDP-glucuronosyltransferase and sulfotransferase were increased by HgCl2 and MeHg, but not by Zuotai and HgS. HgCl2 decreased the expression of organic anion transporter (Oatp1a1), but increased Oatp1a4. Both HgCl2 and MeHg increased the expression of multidrug resistance-associated protein genes (Mrp1, Mrp2, Mrp3, and Mrp4). Zuotai and HgS had little effects on these transporter genes. In conclusion, Zuotai and HgS are different from HgCl2 and MeHg in hepatic drug processing gene expression; suggesting that chemical forms of mercury not only affect their disposition and toxicity, but also affect their effects on the expression of hepatic drug processing genes.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress.

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1µM) and toxic concentrations (5µM) for 24, 48, and 72h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5µM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5nM, while HepG2 cells were insensitive up to 10µM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin.

ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium--Effects of Prochloraz.

Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers.

Sialic acid transporter NanT participates in Tannerella forsythia biofilm formation and survival on epithelial cells.

Tannerella forsythia is a periodontal pathogen implicated in periodontitis. This gram-negative pathogen depends on exogenous peptidoglycan amino sugar N-acetylmuramic acid (NAM) for growth. In the biofilm state the bacterium can utilize sialic acid (Neu5Ac) instead of NAM to sustain its growth. Thus, the sialic acid utilization system of the bacterium plays a critical role in the growth and survival of the organism in the absence of NAM. We sought the function of a T. forsythia gene annotated as nanT coding for an inner-membrane sugar transporter located on a sialic acid utilization genetic cluster. To determine the function of this putative sialic acid transporter, an isogenic nanT-deletion mutant generated by allelic replacement strategy was evaluated for biofilm formation on NAM or Neu5Ac, and survival on KB epithelial cells. Moreover, since T. forsythia forms synergistic biofilms with Fusobacterium nucleatum, co-biofilm formation activity in mixed culture and sialic acid uptake in culture were also assessed. The data showed that the nanT-inactivated mutant of T. forsythia was attenuated in its ability to uptake sialic acid. The mutant formed weaker biofilms compared to the wild-type strain in the presence of sialic acid and as co-biofilms with F. nucleatum. Moreover, compared to the wild-type T. forsythia nanT-inactivated mutant showed reduced survival when incubated on KB epithelial cells. Taken together, the data presented here demonstrate that NanT-mediated sialic transportation is essential for sialic acid utilization during biofilm growth and survival of the organism on epithelial cells and implies sialic acid might be key for its survival both in subgingival biofilms and during infection of human epithelial cells in vivo.

Association of SLCO2B1 Genotypes With Time to Progression and Overall Survival in Patients Receiving Androgen-Deprivation Therapy for Prostate Cancer.

PURPOSE: To validate the association of three previously demonstrated SLCO2B1 germline variants with time to progression (TTP) in patients receiving androgen-deprivation therapy (ADT), and to evaluate if the SLCO2B1 genetic variants impacted overall survival (OS) for prostate cancer (PC). PATIENTS AND METHODS: Three single nucleotide polymorphisms (SNPs), exonic SNP rs12422149 and intronic SNPs rs1789693 and rs1077858, were genotyped in an independent validation cohort of 616 patients with PC who were treated with ADT at the Dana-Farber Cancer Institute from 1996 to 2013. Multivariable Cox proportional hazards regression adjusting for known prognostic factors estimated the association of these genetic variants with TTP and OS in patients receiving ADT. The expression of SLCO2B1 was examined in prostatectomy samples, and the impact of SLCO2B1 expression level on DHEAS (dehydroepiandrosterone sulfate) uptake was evaluated in cell lines. RESULTS: The association between exonic SNP rs12422149 and TTP in patients treated with ADT was confirmed in univariable (P = .019) and multivariable analyses (adjusted hazard ratio, 1.31; 95% CI, 1.00 to 1.72 for GG v AA/AG; P = .049). Because OS had not been previously evaluated, we examined the association in the combined initial and validation cohorts (N = 1,094). The intronic SNP rs1077858 was associated with OS in both univariable (P = .009; Bonferroni's method adjusted P = .027) and multivariable analyses (adjusted hazard ratio, 1.35; 95% CI, 1.07 to 1.71 for GG v AA/AG; P = .012). SLCO2B1 expression in normal prostate tissue and in 22RV1 cells carrying the major allele of SNP rs1077858 was significantly lower than in cells carrying the risk allele. We show in vitro that SLCO2B1 expression levels correlated with DHEAS uptake by PC cells. CONCLUSION: The association of SNP rs1077858 with OS may be a result of differential SLCO2B1 expression and the consequent increased uptake of DHEAS and subsequent resistance to ADT, which, in turn, may contribute to decreased OS.

Expression of steroid sulfated transporters and 3ß-HSD activity in endometrium of women having polycystic ovary syndrome.

Intracrinology mechanism involves the metabolism of steroids in peripheral tissues, such as DHEA, to molecules with estrogenic or androgenic activity. Proliferation rate of endometria from Polycystic Ovary Syndrome women (PCOS) is increased, favoring hyperplasia development. Besides, in endometria from PCOS-women the synthesis of androst-5-ene-3ß,17ß-diol (androstenediol), an estrogenic molecule, is enhanced concomitantly to increased cellular proliferation. DHEA, the major intracrinological precursor, circulates mainly in its sulfated form and requires transporters for cell intake, that belong to the families of organic anion transporting polypeptides (OATP) and organic anion transporters (OAT). The aim of this study was to determine protein levels and activity of sulfated steroid transporters OATP2B1, OATP3A1, OATP4A1 and OAT4 in endometria from control and PCOS-women and to evaluate the activity of the enzyme 3ß-HSD. Levels of transporters were done by RT-PCR (OAT4 only) and Western-blot (WB). Additionally, in primary culture cells stimulated with steroids, protein levels by WB and uptake of tritiated DHEAS, were evaluated; 3ß-HSD activity was assessed using radiolabel substrate. PCOS-endometrium had higher levels of OATP2B1 and OATP4A1 than CE (p<0.05); decreased OATP4A1 levels were found in androstenediol or testosterone-stimulated cells. Accordingly, the entry of DHEAS to cells was lower in cells stimulated with testosterone (p<0.05); 3ß-HSD-activity was similar in control and PCOS-endometria. Therefore, this study describes that steroids can modulate the expression and activity of transporters of OATPs-family in human endometria and that some transporter levels are increased in PCOS-endometria, suggesting a potential role in the pathogenesis of endometrial hyperplasia of these patients.

Functional expression of the 11 human Organic Anion Transporting Polypeptides in insect cells reveals that sodium fluorescein is a general OATP substrate.

Organic Anion Transporting Polypeptides (OATPs), encoded by genes of the Solute Carrier Organic Anion (SLCO) family, are transmembrane proteins involved in the uptake of various compounds of endogenous or exogenous origin. In addition to their physiological roles, OATPs influence the pharmacokinetics and drug-drug interactions of several clinically relevant compounds. To examine the function and molecular interactions of human OATPs, including several poorly characterized family members, we expressed all 11 human OATPs at high levels in the baculovirus-Sf9 cell system. We measured the temperature- and inhibitor-sensitive cellular accumulation of sodium fluorescein and fluorescein-methotrexate, two fluorescent substrates of the OATPs, OATP1B1 and 1B3. OATP1B1 and 1B3 were functional in Sf9 cells, showing rapid uptake (t1/2(fluorescein-methotrexate) 2.64 and 4.16 min, and t1/2(fluorescein) 6.71 and 5.58 min for OATP1B1 and 1B3, respectively) and high-affinity transport (Km(fluorescein-methotrexate) 0.23 and 0.53 µM, and Km(fluorescein) 25.73 and 38.55 µM for OATP1B1 and 1B3, respectively) of both substrates. We found that sodium fluorescein is a general substrate of all human OATPs: 1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, while fluorescein-methotrexate is only transported by 1B1, 1B3, 1A2 and 2B1. Acidic extracellular pH greatly facilitated fluorescein uptake by all OATPs, and new molecular interactions were detected (between OATP2B1 and Imatinib, OATP3A1, 5A1 and 6A1 and estradiol 17-ß-d-glucuronide, and OATP1C1 and 4C1 and prostaglandin E2). These studies demonstrate, for the first time, that the insect cell system is suitable for the functional analysis of the entire human OATP family, and for drug-OATP interaction screening.

Identification and Functional Characterization of a GSH Conjugate Efflux Pathway in the Rat Lens.

PURPOSE: To identify and functionally characterize transporters involved in the release of glutathione (GSH) conjugates from the rat lens. METHODS: Polymerase chain reaction and Western blotting were used to screen for the presence of multidrug resistance-associated protein (Mrp) and organic anion transporting polypeptide (Oatp) isoforms, and immunohistochemistry used to localize Mrp isoforms. To test for Mrp function, lenses were loaded with 5-chloromethylfluorescein diacetate and monochlorobimane to form the fluorescent GSH conjugates glutathione methylfluorescein (GS-MF) and glutathione bimane (GS-B), respectively, and cultured in artificial aqueous humour (AAH) in the presence or absence of MK571, an Mrp-specific inhibitor, or benzbromarone, a nonspecific organic anion transporter inhibitor. Glutathione-MF and GS-B fluorescence were measured in the AAH media and lenses. RESULTS: Multidrug resistance-associated proteins 1, 4, 5, and Oatp1a4 were present at the transcript level, but only Mrp1, 4, and 5 were detected at the protein level. Multidrug resistance-associated proteins 1 and 5 localized to the epithelium and peripheral fiber cells, whereas Mrp4 strongly labeled the nuclei. Glutathione-MF and GS-B efflux was significantly decreased and accumulation in the lens significantly increased in the presence of MK571, indicating that the Mrps are the predominant transporters involved in GSH conjugate release from the lens. Glutathione-B conjugate efflux was further inhibited in the presence of benzbromarone, suggesting that alternative organic anion pathways were involved in mediating GS-B efflux. CONCLUSIONS: Multidrug resistance-associated proteins are present in the lens and may be used to remove endogenous and exogenous compounds from the lens via GSH conjugation. This may represent an important pathway of detoxification required to minimize oxidative stress and maintain lens homeostasis.

Ethnic variability in the expression of hepatic drug transporters: absolute quantification by an optimized targeted quantitative proteomic approach.

Hepatic OATPs 1B1, 1B3 and 2B1, as well as P-gp, play important roles in regulating liver uptake and biliary excretion of drugs. The intrinsic ethnic variability in OATP1B1-mediated hepatic uptake of statins has been proposed to underlie the ethnic variability in plasma exposures of statins between Caucasians and Asians. Using a targeted quantitative proteomic approach, we determined hepatic protein concentrations of OATP1B1, OATP1B3, OATP2B1, P-gp, and PMCA4 (a housekeeping protein) in a panel of human livers (n = 141) and compared protein expression across Caucasian, Asian, African-American, and unidentified donors. Using an optimized protocol that included sodium deoxycholate as a membrane protein solubilizer, the hepatic protein expression levels (mean ± S.D.) of these transporters across all livers were determined to be 15.0 ± 6.0, 16.1 ± 8.1, 4.1 ± 1.3, 0.6 ± 0.2, and 2.4 ± 1.0 fmol/µg of total membrane protein, respectively. The scaling factor was 3.5 mg of total membrane protein in 100 mg of wet liver tissue. OATP1B1 protein expression was significantly associated with the c.388A>G (rs2306283, N130D) single nucleotide polymorphism. When compared across ethnicity, the hepatic expression levels of OATP1B1 and OATP1B3 were unexpectedly higher in Asians relative to Caucasians, suggesting that hepatic OATP expression alone does not explain the increased systemic statin levels in Asians compared with Caucasians. These findings may help improve physiologically based pharmacokinetic modeling to predict statin pharmacokinetic profiles and enable extrapolation of pharmacokinetic data of OATP substrates across ethnic groups.

Schisandrin A and B induce organic anion transporting polypeptide 1B1 transporter activity.

Organic anion transporting polypeptide 1B1 (OATP1B1) is the most important transporter in the organic anion transporting polypeptide family. OATP1B1 plays an important role in the hepatic uptake of many endogenous compounds and xenobiotics, including many clinical drugs. At present, the combinational usage of Chinese traditional herbal medicines and conventional chemical pharmaceuticals may affect the activity of enzymes and transporters activity and cause absorption of their substrates and metabolic changes. In this study, we aimed to investigate the effect of schisandrin A, schisandrin B and tanshinone IIA, which were extracted from medicinal plants, on OATP1B1 activity. HepG2 cells are used as in vitro models for OATP1B1 activity studies. A combination of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tertazolium bromide (MTT) assays, real-time RT-PCR, and transporter activity analysis were employed. We found that schisandrin A and B increased OATP1B1 mRNA levels by 1.81-fold (p < 0.01) and 1.87-fold (p < 0.01) at concentration of 10 µM, respectively. Schisandrin A of 1 µM and 10 µM and schisandrin B of 10 µM significantly increased the uptake of [3H] estrone-3-sulfate (p < 0.05 or p < 0.01). Tanshinone IIA had no effect on the mRNA expression and transport activity of OATP1B1 at nontoxic concentrations. Our study suggests that schisandrin A and B induced OATP1B1 expression and increased its transporter activity in HepG2 cells.

Evidence of estrone-sulfate uptake modification in young and middle-aged rat prostate.

High plasma exposure to estrogens is often associated with prostate cancer. Reducing this phenomenon may present therapeutic benefits. The involvement of estrone sulphate (E1S), the most abundant circulating estrogen in men, has been partially studied in this age-related pathology. To investigate the consequences of plasma E1S overload on blood and prostate sex steroid levels and inflammatory tissue responses, young and middle-aged male rats were treated with E1S with or without steroid sulfatase (STS) inhibitor STX64 for 21 consecutive days. A plasma and prostate tissue steroid profile was determined. STS activity, mRNA expression of E1S organic anion transporting polypeptides (slco1a2, slco2b1, slco4a1) and pro-inflammatory cytokines (Il1-beta, Il6, TNF-alpha) were evaluated in prostate tissue according to age and treatment group. A significant correlation between plasma and prostate steroid levels related to hormone treatment was observed in all rat age groups. However, while the E1S level in prostate tissue increased in middle-aged treated rats (p<0.0001), no significant variation was observed in young treated rats. The protective effect of STX64 during E1S infusion was observed by the maintenance of low free estrogen concentrations in both plasma and tissue. However, this protection was not associated with mRNA expression stability of pro-inflammatory cytokines in older rat prostate. These results suggest that E1S uptake in rat prostate cells increases during aging. Therefore, if a similar phenomenon existed in men, preventively reducing the STS activity could be of interest to limit uptake of estrogens in prostate when high E1S plasma level is assayed.

Solute Carrier Family of the Organic Anion-Transporting Polypeptides 1A2- Madin-Darby Canine Kidney II: A Promising In Vitro System to Understand the Role of Organic Anion-Transporting Polypeptide 1A2 in Blood-Brain Barrier Drug Penetration.

Organic anion-transporting polypeptide (OATP) 1A2 has the potential to be a target for central nervous system drug delivery due to its luminal localization at the human blood-brain barrier and broad substrate specificity. We found OATP1A2 mRNA expression in the human brain to be comparable to breast cancer resistance protein and OATP2B1 and much higher than P-glycoprotein (P-gp), and confirmed greater expression in the brain relative to other tissues. The goal of this study was to establish a model system to explore OATP1A2-mediated transcellular transport of substrate drugs and the interplay with P-gp. In vitro (human embryonic kidney 293 cells stably expressing Oatp1a4, the closest murine isoform) and in vivo (naïve and Oatp1a4 knock-out mice) studies with OATP1A2 substrate triptan drugs demonstrated that these drugs were not Oatp1a4 substrates. This species difference demonstrates that the rodent is not a good model to investigate the active brain uptake of potential OATP1A2 substrates. Thus, we constructed a novel OATP1A2 expressing Madin-Darby canine kidney (MDCK) II wild type and an MDCKII-multidrug resistance protein 1 (MDR1) system using BacMam virus transduction. The spatial expression pattern of OATP1A2 after transduction in MDCKII-MDR1 cells was superimposed to P-gp, confirming apical membrane localization. OATP1A2-mediated uptake of zolmitriptan, rosuvastatin, and fexofenadine across monolayers increased with increasing OATP1A2 protein expression. OATP1A2 counteracted P-gp efflux for cosubstrates zolmitriptan and fexofenadine. A three-compartment model incorporating OATP1A2-mediated influx was used to quantitatively describe the time- and concentration-dependent apical-to-basolateral transcellular transport of rosuvastatin across OATP1A2 expressing the MDCKII monolayer. This novel, simple and versatile experimental system is useful for understanding the contribution of OATP1A2-mediated transcellular transport across barriers, such as the blood-brain barrier.