Tetrabromobisphenol A (TBBPA) Alters ABC Transport at the Blood-Brain Barrier.

Tetrabromobisphenol A (TBBPA, CAS No. 79-94-7) is a brominated flame retardant used in 90% of epoxy coated circuit boards. Exposures to TBBPA can induce neurotoxicity and disrupt MAPK, estrogen, thyroid, and PPAR-associated signaling pathways. Because these pathways also regulate transporters of the central nervous system barriers, we sought to determine the effect of TBBPA on the expression and activity of 3 ATP binding cassette (ABC) transporters of the blood-brain barrier (BBB). Using a confocal based assay, we measured the ex vivo and in vivo effects of TBBPA on P-glycoprotein (P-gp), breast cancer resistant protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) transport activity in rat brain capillaries. Our rationale for using a rat model was based on tissue availability, ease of handling, and availability of historical TBBPA toxicokinetic data. We found that TBBPA (1-1000 nM) exposure had no significant effect on multidrug resistance-associated protein 2 transport activity in either sex, suggesting TBBPA does not compromise the physical integrity of the BBB. However, low concentrations of TBBPA (1-100 nM) significantly decreased breast cancer resistant protein transport activity in both sexes. Additionally, TBBPA exposures (1-100 nM), elicited a sex-dependent response in P-gp transport: increasing transport activity in males and decreasing transport activity in females. All TBBPA dependent changes in transport activity were dose- and time-dependent. Inhibitors of either transcription or translation abolished the TBBPA dependent increases in male P-gp transport activity. Western blot and immunofluorescent assays confirmed the TBBPA dependent P-gp increases expression in males and decreases in females. Antagonizing PPAR-γ abolished the TBBPA dependent increases in males but not the decreases in females. However, the decreases in female P-gp transport were blocked by an ER-α antagonist. This work indicates that environmentally relevant concentrations of TBBPA (1-100 nM) alter ABC transporter function at the BBB. Moreover, permeability changes in the BBB can alter brain homeostasis, hinder central nervous system drug delivery, and increase the brain's exposure to harmful xenobiotic toxicants.

Therapies targeting exogenous cholesterol uptake: new insights and controversies.

Exogenous cholesterol uptake involves a complex process in the intestines for the absorption of cholesterol and bile acids. This process is regulated by intestinal nuclear transcription factors such as LXR that affect sterol transporters NPC1L1, ABCG5/G8, and ABCG1, and enzymes such as ACAT-2. Plant sterol/stanols, ezetimibe, and bile acid sequestrants have a variety of effects on these various transporters, and new insights into their mechanism(s) of action have provided a plethora of exciting targets for metabolic diseases, dyslipidemia, and atherosclerosis.

Multidrug resistance proteins restrain the intestinal absorption of trans-resveratrol in rats.

trans-Resveratrol, a natural antioxidant, has been described as a nutraceutic compound with important beneficial effects on health, but its low oral bioavailability hinders its therapeutic activity. Here, we studied the mechanisms of apical transport of trans-resveratrol in enterocytes and the role of ATP-binding cassette (ABC) transporters in the secretion of resveratrol glucuronide and sulfate resulting from the rapid intracellular metabolism. An intestinal perfusion method with recirculation in vivo was used in rats. Jejunal loops were perfused with increasing concentrations of trans-resveratrol and results showed that its uptake occurs by simple diffusion without the participation of a mediated transport. The apparent diffusion constant was 8.1 +/- 0.3 microL/(5 min.mg dry weight). The glycoprotein-P (Pgp, ABCB1), multidrug resistance-associated protein 2 (MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2) located in the apical membrane of enterocytes were investigated using specific inhibitors. The Pgp inhibitors verapamil (5 micromol/L) and cyclosporin A (5 micromol/L) did not affect the efflux of trans-resveratrol and its conjugates. The MRP2 inhibitors probenecid (2 mmol/L) and MK571 (10 micromol/L) reduced the efflux of glucuronide by 61 and 55%, respectively, and of sulfate by 43 and 28%, respectively. The BCRP inhibitor Ko143 (0.5 micromol/L) decreased the secretion of glucuronide by 64% and of sulfate by 46%. Our experiments identify MRP2 and BCRP as the 2 apical transporters involved in the efflux of resveratrol conjugates.

Expression of MDR1, MRP2 and BCRP mRNA in tissues of turkeys.

MDR1, MRP2 and BCRP are members of the superfamily of ABC membrane transporters that export a large variety of structurally diverse substances out of the cell, hence being an integral part of various biological barriers. Here we report for the first time the tissue distribution of these ABC efflux transporters in the gastrointestinal tract (crop, proventriculus, duodenum, proximal and distal jejunum, ileum, caecum, colon) as well as in liver, kidney, lung, brain, adrenal gland, ovaries, oviduct and testes in BUT9 turkeys. MDR1 and BCRP mRNA expression was detected in all tissue samples, and the highest levels were measured in the small intestines. The tissue distribution of MRP2 mRNA was less consistent and some tissues seemed to lack any significant expression. Moreover, in consideration of previous findings suggesting that fluoroquinolones are substrates and modulators of ABC transporters, the effect of orally administered danofloxacin mesylate on the levels of MDR1, MRP2 and BCRP mRNA expression was investigated. Danofloxacin treatment resulted in a significant up-regulation of the measured transporters at the transcriptional level in the upper part of gastro-intestinal tract, liver and kidneys as well as in barrier-protected organs, such as the brain. However, despite this significant increase in the transcription levels, the pharmacokinetic parameters after repeated application of danofloxacin mesylate were not significantly altered.

The effect of ABCG2 V12M, Q141K and Q126X, known functional variants in vitro, on the disposition of lamivudine.

AIMS: To evaluate the effects of three ABCG2 variants (Q141K, V12M and Q126X), which are known to have altered transport properties in vitro, on the disposition of lamivudine in healthy subjects. METHODS: To evaluate whether lamivudine is a substrate of ABCG2, intracellular accumulation and vectorial transport of 3H-lamivudine were determined in MDCK-ABCG2 cells. The pharmacokinetic parameters of lamivudine were compared among subjects with four different ABCG2 genotypes, including wild type (seven subjects), K141/K141 (six subjects), Q126/Stop126 (four subjects) and M12/M12 (five subjects) after a single oral dose of 100 mg lamivudine. RESULTS: The intracellular accumulation of lamivudine in MDCK-ABCG2 cells was significantly lower than that in MDCK-mock cells, but fumitremorgin C reversed the intracellular lamivudine concentration to that of MDCK-mock cells. The ABCG2-mediated transport of lamivudine was saturable and the values of Km and Vmax were 216.5 +/- 58 microm and 20.42 +/- 2.9 nmol h(-1) per 10(6) cells, respectively. After lamivudine administration to healthy subjects, the AUC of lamivudine showed no difference among subjects with different ABCG2 genotypes; 2480 +/- 502, 2207 +/- 1019, 2422 +/- 239, 2552 +/- 698 ng h(-1) ml(-1) for wild type, K141/K141, Q126/Stop126 and M12/M12 genotype, respectively (P = 0.85). The estimated 95% confidence intervals for the mean difference between K141/K141, Q126/Stop126, M12/M12 and wild as reference were (-1053, 507), (-555, 439) and (-552, 696), respectively. No other pharmacokinetic parameters were estimated to be significantly different among four different ABCG2 genotypes tested. CONCLUSIONS: Lamivudine appeared to be a substrate of ABCG2 in vitro, but the disposition of lamivudine was not significantly influenced by known in vitro functional variants of ABCG2, Q141K, V12M and Q126X in healthy subjects.

Lack of interactions between breast cancer resistance protein (bcrp/abcg2) and selected antiepileptic agents.

PURPOSE: Recent studies have indicated constitutive expression of efflux transporter, breast cancer resistance protein (BCRP, ABCG2), in endothelial cells of the blood-brain barrier (BBB). In epileptogenic brain tumors such as ganglioma, astrocytoma, anaplastic astrocytomas, or glioma multiforme, strong expression of BCRP in the microvasculature of the BBB was observed. Therefore it was hypothesized that this phenomenon could critically influence the bioavailability of drugs in these tumors and potentially contribute to the failure of antiepileptic treatment. The aim of this study was to test whether some commonly used antiepileptic drugs (AEDs) are substrates transported by human BCRP. In particular, we focused on phenobarbital, phenytoin, ethosuximide, primidone, valproate, carbamazepine, clonazepam, and lamotrigine. Furthermore, the inhibitory potency of these AEDs to BCRP was examined. METHODS: To study substrate affinity of tested AEDs to BCRP, transport experiments were performed in epithelial BCRP-expressing MDCKII-BCRP and MDCKII-parent cell lines cultured on microporous membrane. For detection of inhibitory potency of AEDs to BCRP, accumulation assays were carried out in MEF3.8-BCRP cells with known BCRP substrates, BODIPY FL prazosin and mitoxantrone. RESULTS: No obvious interactions of tested AEDs with BCRP transporter were observed. Therefore these drugs in relevant therapeutic concentrations are neither substrates nor inhibitors of BCRP. CONCLUSIONS: Based on our in vitro data we can conclude that resistance to treatment with the tested AEDs probably is not caused by the overexpression of BCRP in the BBB of epileptogenic brain tumors.

RLIP76 transports vinorelbine and mediates drug resistance in non-small cell lung cancer.

Vinorelbine (Navelbine), an amphiphilic semisynthetic Vinca alkaloid, has displayed superior activity and decreased resistance in the treatment of advanced non-small cell lung cancer (NSCLC) compared with other members of its class. Recently, vinorelbine and cisplatin combination chemotherapy has been shown for the first time to confer a significant survival advantage in early-stage lung cancer after surgical therapy. The biological mechanisms underlying the differential response of NSCLC to cytocidal activity of vinorelbine have yet to be elucidated. Our recent findings indicate a role of RLIP76, a non-ATP binding cassette transport protein, in catalyzing the ATP-dependent efflux of structurally and functionally unrelated chemotherapeutic agents such as doxorubicin and vinblastine in NSCLC. Present studies were conducted to assess whether RLIP76 mediates vinorelbine transport and resistance. Here we show that RLIP76 catalyzes the transport of vinorelbine in a saturable manner with respect to vinorelbine (K(m) 75 nmol/L) and ATP (K(m) = 3.4 mmol/L). Three-fold overexpression of RLIP76 in NSCLC and SCLC confers increased resistance to cytotoxicity. RLIP76 overexpression causes a sustained intracellular decrease in vinorelbine concentration because of increased efflux, and anti-RLIP76 antibodies sensitize lung cancer cells to vinorelbine by inhibiting its efflux. These studies for the first time show that RLIP76 mediates vinorelbine transport and is capable of conferring drug accumulation defect and resistance to lung cancer cells.

The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability.

Many orally administered drugs must overcome several barriers before reaching their target site. The first major obstacle to cross is the intestinal epithelium. Although lipophilic compounds may readily diffuse across the apical plasma membrane, their subsequent passage across the basolateral membrane and into blood is by no means guaranteed. Efflux proteins located at the apical membrane, which include P-glycoprotein (Pgp; MDR1) and MRP2, may drive compounds from inside the cell back into the intestinal lumen, preventing their absorption into blood. Drugs may also be modified by intracellular phase I and phase II metabolising enzymes. This process may not only render the drug ineffective, but it may also produce metabolites that are themselves substrates for Pgp and/or MRP2. Drugs that reach the blood are then passed to the liver, where they are subject to further metabolism and biliary excretion, often by a similar system of ATP-binding cassette (ABC) transporters and enzymes to that present in the intestine. Thus a synergistic relationship exists between intestinal drug metabolising enzymes and apical efflux transporters, a partnership that proves to be a critical determinant of oral bioavailability. The effectiveness of this system is optimised through dynamic regulation of transporter and enzyme expression; tissues have a remarkable capacity to regulate the amounts of protein both at transcriptional and post-transcriptional levels in order to maintain homeostasis. This review addresses the progress to date on what is known about the role and regulation of drug efflux mechanisms in the intestine and liver.

Effect of organic isothiocyanates on breast cancer resistance protein (ABCG2)-mediated transport.

PURPOSE: To investigate the effect of organic isothiocyanates (ITCs), dietary compounds with chemopreventive activity, on breast cancer resistance protein (BCRP)-mediated transport. METHODS: The effect of 12 ITCs on the cellular accumulation of mitoxantrone (MX) was measured in both BCRP-overexpressing and BCRP-negative human breast cancer (MCF-7) and large cell lung carcinoma (NCI-H460) cells by flow cytometric analysis. The ITCs showing activity in MX accumulation were examined for their effect on MX cytotoxicity, and the intracellular accumulation of radiolabeled phenethyl isothiocyanate (PEITC) was measured in both BCRP-overexpressing and BCRP-negative NCI-H460 cells. RESULTS: ITCs significantly increased MX accumulation and reversed its cytotoxicity in resistant cells, but had a small or no effect in sensitive cells. The effects of ITCs on MX accumulation and cytotoxicity were ITC-concentration dependent. Significant increases in MX accumulation were observed at ITC concentrations of 10 or 30 microM, and significant reversal of MX cytotoxicity was generally observed at ITC concentrations of 10 microM. Intracellular accumulation of radiolabeled PEITC in BCRP-overexpressing cells was significantly lower than that in BCRP-negative cells and was increased significantly by the BCRP inhibitor fumitremorgin C (FTC). CONCLUSIONS: Certain ITCs are BCRP inhibitors and PEITC and/or its cellular metabolite(s) may represent BCRP substrates, suggesting the potential for diet-drug interactions.

Effect of mdr1a P-glycoprotein gene disruption, gender, and substrate concentration on brain uptake of selected compounds.

PURPOSE: This study assessed the influence of mdr1a P-glycoprotein (P-gp) gene disruption, gender and concentration on initial brain uptake clearance (Clup) of morphine, quinidine and verapamil. METHODS: Clup of radiolabeled substrates was determined in P-gp-competent and deficient [mdr1a(-/-)] mice by in situ brain perfusion. Brain:plasma distribution of substrates after i.v. administration was determined in both strains. RESULTS: Genetic disruption of mdr1a P-gp resulted in 1.3-, 6.6- and 14-fold increases in Clup for morphine, verapamil and quinidine, respectively. With the exception of small differences for verapamil, gender did not affect Clup. Saturable transport of verapamil and quinidine was observed only in P-gp-competent mice, with apparent IC50 values for efflux of 8.6 +/- 2.3 microM and 36 +/- 2 microM, respectively. Verapamil Clup was approximately 50% higher in mdr1a(+/-) vs. mdr1a(+/+) mice; no such difference was observed for quinidine. In P-gp-competent mice, uptake of verapamil and quinidine was unaffected by organic vehicles. Plasma decreased VER Clup to a greater extent in the presence of P-gp. The influence of P-gp in situ was lower than, but correlated with, the effect in vivo. CONCLUSIONS: P-gp decreases Clup of morphine, verapamil and quinidine in situ with little or no influence of gender, but this effect cannot fully account for the effects of P-gp in vivo. P-gp is the only saturable transport mechanism for verapamil and quinidine at the murine blood-brain barrier. The influence of protein binding on Clup may be enhanced by P-gp-mediated efflux.

ABC1, an ATP binding cassette transporter required for phagocytosis of apoptotic cells, generates a regulated anion flux after expression in Xenopus laevis oocytes.

The ATP binding cassette transporter ABC1 is a 220-kDa glycoprotein expressed by macrophages and required for engulfment of cells undergoing programmed cell death. Since members of this family of proteins such as P-glycoprotein and cystic fibrosis transmembrane conductance regulator share the ability to transport anions, we have investigated the transport capability of ABC1 expressed in Xenopus oocytes using iodide efflux and voltage-clamp techniques. We report here that ABC1 generates an anion flux sensitive to glibenclamide, sulfobromophthalein, and blockers of anion transporters. The anion flux generated by ABC1 is up-regulated by orthovanadate, cAMP, protein kinase A, and okadaic acid. In other ABC transporters, mutating the conserved lysine in the nucleotide binding folds was found to severely reduce or abolish hydrolysis of ATP, which in turn altered the activity of the transporter. In ABC1, replacement of the conserved lysine 1892 in the Walker A motif of the second nucleotide binding fold increased the basal ionic flux, did not alter the pharmacological inhibitory profile, but abolished the response to orthovanadate and cAMP agonists. Therefore, we conclude that ABC1 is a cAMP-dependent and sulfonylurea-sensitive anion transporter.

Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP). In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry. The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines. R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance. Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP. In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance. The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP. Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies.