BCRP mRNA Levels Research Articles

Downregulation of BCRP (ABCG2) in Placenta of Rat Model of Preeclampsia

BACKGROUNDRecent studies in our laboratory demonstrated downregulation of breast cancer resistance protein (BCRP/ABCG2) in placenta obtained from women with preeclampsia (PE). Pregnant women with PE are often prescribed medications to manage disease progression and prevent adverse outcomes. Many of these drugs, as well as other clinically relevant therapeutics are substrates for BCRP. BCRP is highly expressed in placenta and plays an important role in preventing xenobiotics from crossing the placenta and entering the fetal compartment. Thus, the impact of PE and BCRP downregulation on fetal drug exposure needs to be examined. As studies in pregnant women are often not feasible, use of preclinical models is an important approach. Thus, our objective was to characterize transporter changes in an endotoxin rat model of PE to determine its utility and predictive value for future drug disposition studies. This frequently utilized PE model is associated with maternal immune activation and induction of PE‐like symptoms including high blood pressure, proteinuria and pathology which are characteristic of human disease.METHODSPregnant rats were injected intraperitoneally with 10 μg/kg of bacterial endotoxin (LPS) on GD13, followed by 40 μg/kg LPS daily until GD 16. Controls were injected with sterile saline. Urine was collected daily. Dams were sacrificed on GD17 or GD18 for collection of maternal plasma, placentas, and fetuses (n=7‐8/group/day). Plasma levels of TNF‐α and IL‐6 were quantified by ELISA Kit. Tissues were analyzed for the expression of transporters by using qPCR and western blot. Urine was analyzed using creatinine colorimetric and protein assays.RESULTSConsistent with previous reports, the urinary ratio of protein/creatinine was significantly elevated in PE dams on GDs 16‐18, demonstrating proteinuria. As compared to controls, maternal plasma concentrations and placental mRNA levels of the pro‐inflammatory cytokines, TNF‐α and IL‐6, were significantly higher in the PE group on GD17 and GD18. The mRNA levels of BCRP was significantly decreased by 40‐60 % in placenta of PE dams on GD17 and GD18 along with a corresponding decrease in protein expression on GD18. Significantly lower mRNA levels of Abcb1a, Abcb1b and Slco2b1 were observed in the PE group. PE was also associated with significantly decreased transcript levels of Bcrp, Abcb1a and Abc1b in fetal brains.CONCLUSIONOur data demonstrates that the endotoxin PE rat model exhibits proteinuria and immune activation consistent with human disease. Importantly, our observations of decreased placenta expression of BCRP in this PE model is consistent with findings in humans. This indicates that this preclinical model may serve as a useful translational tool to examine the impact of PE on the disposition and fetal exposure of BCRP drug substrates.

Effect of capsaicin on breast cancer resistance protein (Bcrp/Abcg2) and pharmacokinetics of probe substrates in rats

1. Breast cancer resistance protein (BCRP/ABCG2 in human, Bcrp/Abcg2 in rat), a member of the ATP-binding cassette (ABC) transporter family, acts as an efflux pump for xenobiotics, with ability to transport various drugs out of cells. Capsaicin may have the potential to modulate the function of Bcrp transport. This study was to evaluate the effects of capsaicin on the pharmacokinetics of sulfasalazine, a Bcrp substrate, in rats and investigate the mechanism of this food-drug interaction. 2. The rats were pretreated with 5% carboxymethylcellulose sodium (vehicle), capsaicin (3, 8, 25 mg/kg) and cyclosporine A (10 mg/kg) by gastric gavage for 7 days. On day 7, blood, liver and intestine samples were collected after sulfasalazine administered. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to study the effects of capsaicin on the pharmacokinetics of sulfasalazine in rats. RT-PCR and western blotting were used to study the mechanism in biomolecules in rats, respectively. 3. Compared with vehicle group, AUC0-∞ of sulfasalazine in rats were increased by 1.5 folds, 1.6 folds and 1.7 folds in 3, 8 and 25 mg/kg/d capsaicin pretreated groups. At the same time, the CL/F in rats were decreased by 33%, 38% and 42% in the three groups. In addition, we found Bcrp mRNA levels and protein expressions in rat livers and intestines were decreased in 3, 8 and 25 mg/kg/d capsaicin-treated groups. 4. Our study demonstrated that long-term ingestion of capsaicin significantly enhanced the AUC of sulfasalazine involved down-regulate Bcrp gene and protein expression in rat liver and intestine.

Effect and molecular mechanism research of Astragalus membranaceus on inhibiting intestinal absorption of six alkaloids of Aconitum carmichaelii in spleen deficiency rats

ObjectiveTo investigate the effect and the mechanism of Astragalus membranaceus (Huangqi in Chinese, HQ) extract on the intestinal absorption of six alkaloids of Aconitum carmichaelii (Fuzi in Chinese, FZ) in rats with spleen deficiency and provide novel insights into the application of HQ on modulating intestinal barrier. MethodsFour-week-old male Sprague-Dawley rats were fed with Xiaochengqi Decoction to induce the spleen deficiency model for 40 d. Single-pass intestinal perfusion model were used to study the effects of HQ extract on the absorption of alkaloids. Protein expression and mRNA levels of MRP2 and BCRP and tight junction proteins (TJ, including Claudin-1, Occludin and ZO-1) were measured using Western blot and real-time PCR, respectively. The location and expression of TJ protein was also investigated by the immunofluorescence method. ResultsCompared with the normal group, the protein expression of MRP2, BCRP and TJ proteins in the model group were significantly down-regulated. After oral administration of HQ, the alkaloid absorption in intestinal villi was inhibited, MRP2, BCRP and TJ proteins were up-regulated, the green fluorescence staining of Claudin-1, Occludin, and ZO-1 was enhanced, and a thick layer of mucus was deposited on the surface of the epithelium of the intestinal cavity. ConclusionHQ as an intestinal barrier modulator improves the physiological changes of the intestinal environment of spleen deficiency to reduce the absorption of toxic components, leading to a decrease in the absorption of drug-like molecules.

Effects of carfilzomib alone and in combination with cisplatin on the cell death in cisplatin-sensitive and cisplatin-resistant ovarian carcinoma cell lines.

Previous studies on the efficacy of platinum-based drugs and selective inhibitors of proteasome have revealed promising outcomes. This study is aimed to evaluate the effects of the combination of cisplatin and carfilzomib on the cell death induction and drug efflux transporters expression in cisplatin-sensitive (A2780s) and cisplatin-resistant (A2780cp) ovarian cancer cells lines. MTT cytotoxic assay was conducted to determine the cytotoxicity. Drug interactions were analyzed based on Chou-Talalay's principles and real-time PCR analysis was performed to determine possible alterations in mRNA levels of MRP1 and BCRP. A2780s cells were more susceptible to both cisplatin and carfilzomib while analyses of drug interactions between the two agents showed synergistic effects in all affected fractions of drug-treated A2780s and A2780cp cells (CI<0.9) with the combination indices being significantly lower in A2780cp cells (p < 0.01). We also found that although mRNA levels of BCRP and MRP1 were significantly altered in both cells exposed to each drug alone, only the combination regimen was able to significantly reduce the mRNA levels of these genes in A2780cp cells (p<0.001). This combination might be a potential strategy for suppressing cell growth via downregulating the drug efflux transporters expression, especially in cisplatin-resistant ovarian cancer cells (Fig. 3, Ref. 45).

Histone Modification is a Novel Epigenetic Mechanism to Up‐Regulate Human Blood‐Brain Barrier Transporters

Multidrug Resistance Protein 1 (MDR1, ABCB1) and the Breast Cancer Resistance Protein (BCRP, ABCG2) expressed at the blood‐brain barrier (BBB) are key efflux transporters that extrude chemicals from the BBB and regulate the efficacy and/or toxicity of chemicals in the brain. Prior studies in cancer cells have pointed to the ability of histone deacetylase (HDAC) inhibitors to modulate the expression and function of MDR1 and BCRP. However, whether or not such regulation occurs at the BBB is not known. Here we sought to test whether HDAC inhibitors could potentially alter expression and function of MDR1 and BCRP at the BBB. To test this, we treated immortalized human brain capillary endothelial (hCMEC/D3) cells, a model of the BBB, with six different HDAC inhibitors, valproic acid (VPA), sodium butyrate (NaB), romidepsin, apicidin, suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA), and assessed for expression and function of MDR1 and BCRP. HDAC inhibition following treatment was confirmed by increased levels of acetylated histone H3 protein. After 12 h of treatment, VPA, apicidin, SAHA, and TSA up‐regulated MDR1 mRNA levels between 50% and 200%. All six HDAC inhibitors significantly induced BCRP mRNA levels between 100% and 270%. Similarly, the protein expression of MDR1 and BCRP transporters was up‐regulated about two‐fold at 24 h. Enhanced MDR1 expression corresponded with reduced intracellular accumulation of the substrate rhodamine 123. Collectively, these results demonstrate that HDAC inhibitors up‐regulate MDR1 and BCRP transporters at the BBB by modifying histone acetylation. The clinical use of HDAC inhibitors may enhance efflux transporter activity at the BBB and restrict access of xenobiotics to the brain.Support or Funding InformationSupported by R01ES021800, P30ES005022.

Effect of Subchronic Intravenous Morphine Infusion and Naloxone-Precipitated Morphine Withdrawal on P-gp and Bcrp at the Rat Blood–Brain Barrier

Chronic morphine regimen increases P-glycoprotein (P-gp) and breast cancer-resistance protein (Bcrp) expressions at the rat blood–brain barrier (BBB) but what drives this effect is poorly understood. The objective of this study is to assess subchronic continuous morphine infusion and naloxone-precipitated morphine withdrawal effects on P-gp/Bcrp contents and activities at the rat BBB. Rats were treated either with (i) a continuous i.v. morphine for 120 h, (ii) escalating morphine dosing (10-40 mg/kg, i.p., 5 days), (iii) a chronic morphine regimen (10 mg/kg s.c., 5 days) followed by a withdrawal period (2 days) and treatment for 3 additional days. Animal behavior was assessed after naloxone-precipitated withdrawal (1 mg/kg, s.c.). P-gp/Bcrp expressions and activities were determined in brain microvessels by qRT-PCR, Western blot, UHPLC–MS/MS, and in situ brain perfusion of P-gp or Bcrp substrates. Results show continuous i.v. morphine did not change P-gp/Bcrp protein levels in rat brain microvessels, whereas naloxone-precipitated withdrawal after escalating or chronic morphine dose regimen increased Mdr1a and Bcrp mRNA levels by 1.4-fold and 2.4-fold, respectively. Conversely, P-gp/Bcrp protein expressions remained unchanged after naloxone administration, and brain uptake of [3H]-verapamil (P-gp) and [3H]-mitoxantrone (Bcrp) was not altered. The study concludes subchronic morphine infusion and naloxone-precipitated morphine withdrawal have poor effect on P-gp/Bcrp levels at the rat BBB.

Organic Cation Transporter/Solute Carrier Family 22a is Involved in Drug Transfer into Milk in Mice

Drug transfer into milk is a general concern during lactation. So far, breast cancer resistance protein (Bcrp) is the only transporter known to be involved in this process, whereas participation of other transporters remains unclear. We investigated the importance of organic cation transporter (Oct) in drug transfer into milk in mice. The mammary glands of lactating versus nonlactating FVB strain mice revealed elevated mRNA levels of Oct1 and Bcrp, whereas Oct2 and Oct3 mRNA levels were decreased. Specific uptake of cimetidine, acyclovir, metformin, and terbutaline was observed in human embryonic kidney 293 cells transfected with murine Oct1 or Oct2. The milk‐to‐plasma concentration ratio (M/P) values of cimetidine and acyclovir were significantly decreased in Bcrp knockout and Oct1/2 double‐knockout (DKO) mice compared with control FVB mice, whereas the M/P values of terbutaline and metformin were significantly decreased in Oct1/2 DKO mice alone. These are the first to suggest that Oct1 might be involved in secretory transfer of substrate drugs into milk.

Age-related changes in mRNA levels of hepatic transporters, cytochrome P450 and UDP-glucuronosyltransferase in female rats.

Hepatic transporters and metabolic enzymes affect drug pharmacokinetics. Limited information exists on the alteration in mRNA levels of hepatic transporters and metabolic enzymes with aging. We examined the effects of aging on the mRNA levels of representative hepatic drug transporters and metabolic enzymes by analyzing their levels in 10-, 30- and 50-week-old male and female rats. Levels of mRNA of drug transporters including multidrug resistance protein (Mdr)1a, multidrug resistance-associated protein (Mrp)2, breast cancer resistance protein (Bcrp) and organic anion-transporting polypeptide (Oatp)1a1, and the metabolic enzymes cytochrome P450 (CYP)3A1, CYP3A2 and UDP-glucuronosyltransferase (UGT)1A1 were analyzed using real-time reverse transcriptase polymerase chain reaction. The mRNA levels of transporters in male rats did not decrease with age, while the mRNA levels of Bcrp and Oatp1a1 in female rats decreased with age. The mRNA levels of CYP3A1 and CYP3A2 in male rats were higher than those in female rats. The mRNA levels of metabolic enzymes decreased with age in female but not male rats. In particular, the mRNA levels of UGT1A1 in 10-week-old female rats were higher than those in male rats. mRNA expression of hepatic transporters and metabolic enzymes are more susceptible to aging in female than male rats. The age-related decreases in the mRNA levels of Bcrp, Oatp1a1, CYP3A1 and CYP3A2 in female rats may affect the metabolism and transport of substrates. This study showed that aging affected the mRNA expression of hepatic transporters and metabolic enzymes in rats.

Induction of P‐glycoprotein and Bcrp at the rat blood–brain barrier following a subchronic morphine treatment is mediated through NMDA/COX‐2 activation

Subchronic morphine treatment induces P-glycoprotein (P-gp) up-regulation at the blood-brain barrier. This study investigates the rate and extent to which P-gp and breast cancer-resistance protein (Bcrp) increase at the rat blood-brain barrier following subchronic morphine treatment. Rats were given increasing doses of morphine (10-40 mg/kg) or saline i.p. twice daily for 5 days. The brain cortex large vessels and microvessels were then mechanical isolated 6, 9, 12, 24, and 36 h after the last injection. The gene and protein expression of P-gp and Bcrp in morphine-treated and control rats were compared by qRT-PCR and western blotting. The levels of Mdr1a and Bcrp mRNAs were not significantly modified 6 h post morphine, but the Mdr1a mRNA increased 1.4-fold and Bcrp mRNA 2.4-fold at 24 h. P-gp and Bcrp protein expression in brain microvessels was unchanged 6 h post morphine and increased 1.5-fold at 24 h. This effect was more pronounced in large vessels than in microvessels. However, extracellular morphine concentrations of 0.01-10 μM did not modify the expressions of the MDR1 and BCRP genes in hCMEC/D3 human endothelial brain cells in vitro. MK-801 (NMDA antagonist) and meloxicam (cyclo-oxygenase-2 inhibitor) given after morphine treatment completely blocked P-gp and Bcrp up-regulation. Interestingly, misoprostol and iloprost, two well-known agonists of prostaglandin E2 receptors induced both MDR1 and BCRP mRNA levels in hCMEC/D3. Thus, morphine does not directly stimulate P-gp and Bcrp expression by the brain endothelium, but glutamate released during morphine withdrawal may do so by activating the NMDA/cyclo-oxygenase-2 cascade.

Effects of Chronic Renal Failure on Brain Drug Transporters in Rats

Studies demonstrated that chronic renal failure (CRF) affects the expression and activity of intestinal, hepatic, and renal drug transporters. Such drug transporters are expressed in brain cells and at the blood-brain barrier (BBB), where they limit the entry and distribution of drugs in the brain. Perturbations in brain drug transporter equilibrium by CRF could lead to central drug toxicity. This study evaluates how CRF affects BBB drug transporters using a 5/6 nephrectomized rat model. Protein and mRNA expression of influx transporters [organic anion-transporting polypeptide (Oatp), organic anion transporter (Oat)] and efflux transporters [P-glycoprotein (P-gp), multidrug resistance-related protein (Mrp), breast cancer resistance protein (Bcrp)] were measured in CRF and control rat brain. Intracerebral accumulation of radiolabeled benzylpenicillin, digoxin, doxorubicin, and verapamil was used to evaluate BBB drug permeability. Protein expression of the transporters was evaluated in rat brain endothelial cells (RBECs) and astrocytes incubated with control and CRF rat serum. We demonstrated significant decreases (30-50%) in protein and mRNA levels of Bcrp, Mrp2 to -4, Oat3, Oatp2 and -3, and P-gp in CRF rat brain biopsies, as well as in astrocytes and RBECs incubated with CRF serum. These decreases did not correlate with in vivo changes because BBB permeability of benzylpenicillin was decreased by 30% in CRF rats, whereas digoxin, doxorubicin, and verapamil permeabilities were unchanged. It thus seems that even with decreased drug transporters, BBB integrity and function is conserved in CRF.

Regulation mechanism of breast cancer resistance protein by toremifene to reverse BCRP-mediated multidrug resistance in breast cancer cells

To explore the regulation mechanism of the reversal of breast cancer resistance protein-mediated multidrug resistance by toremifene. Two recombinant plasmids (pcDNA3-promoter-BCRP and pcDNA3-CMV-BCRP) were designed to express the wild-type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a CMV promoter as control, respectively. Two recombinant plasmids were transfected into ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines. Four kinds of BCRP expressing cell lines of MCF-7/Promoter-BCRP, MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP were established in which BCRP was promoted by the BCRP promoter and a CMV promoter as control, respectively. The drug resistant cells were treated with toremifene. Then RT-PCR, Western blot, mitoxantrone efflux assays and cytotoxicity assay were performed to detect the reversal function of BCRP by toremifene on the drug resistance cell lines. Toremifene significantly downregulated BCRP mRNA levels in a dose-dependent manner in ERα-positive MCF-7/Promoter-BCRP cells than that of untreated control cells. In MCF-7/Promoter-BCRP cells, toremifene at the dose of 0.1, 1 and 10 µmol/L decreased BCRP mRNA expression by 29.5% (P < 0.05), 68.1% (P < 0.01) and 97.4% (P < 0.01), respectively. After being treated with toremifene and 17β-estradiol, the BCRP mRNA level in MCF-7/Promoter-BCRP cells was 64.2% ± 1.3%, significantly higher than that of toremifene treatment control cells (3.8% ± 0.2%,P < 0.01). Furthermore, the effect of toremifene on BCRP protein is similar in BCRP mRNA. Toremifene obviously increased the mitoxantrone fluorescence intensity and decreased the efflux activity by 47.3% (P < 0.05) in MCF-7/promoter-BCRP cells when compared with the untreated control, whereas intracellular accumulation of mitoxantrone obviously decreased and the efflux activity increased by 61.5% were observed in combination with 17β-estradiol when compared with toremifene treatment alone. The results therefore suggested that toremifene reversed mitoxantrone resistance in MCF-7/Promoter-BCRP cells. However, in MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP cells, toremifene or in combination with 17β-estradiol did not affect intracellular mitoxantrone uptake. Taken together, our findings indicate that expression of BCRP is downregulated by toremifene, via a novel transcriptional mechanism which might be involved in the ERE of BCRP promoter through ER-mediated to inactivate the transcription of BCRP gene.

Decreased Hepatic Breast Cancer Resistance Protein Expression and Function in Multidrug Resistance-Associated Protein 2-Deficient (TR−) Rats

Multidrug resistance-associated protein (Mrp) 2-deficient (TR(-)) Wistar rats have been used to elucidate the role of Mrp2 in drug disposition. Decreased breast cancer resistance protein (Bcrp) levels were reported in sandwich-cultured hepatocytes (SCH) from TR(-) rats compared with those from wild-type (WT) rats. This study was designed to characterize hepatic Bcrp expression and function in TR(-) rats, using nitrofurantoin and pitavastatin as substrates. Bcrp was knocked down by RNA interference in rat SCH. Antibody BXP53, but not BXP21, specifically detected Bcrp knockdown in SCH. Bcrp protein levels were decreased markedly in TR(-) but not Mrp2-deficient Sprague-Dawley [Eisai hyperbilirubinemic rats (EHBR)] rats. Bcrp mRNA levels were decreased significantly in TR(-) livers as determined by TaqMan real-time reverse transcriptase-polymerase chain reaction. Biliary excretion of nitrofurantoin, a specific Bcrp substrate, was decreased significantly in SCH and isolated perfused livers from TR(-) rats compared with those from WT controls, indicating that hepatic Bcrp function is decreased in TR(-) rats. In Bcrp knockdown SCH, the biliary excretion index and in vitro biliary clearance of pitavastatin were decreased significantly to ∼ 58 and ∼ 52% of control, respectively, indicating that Bcrp plays a role in pitavastatin biliary excretion. Pitavastatin biliary excretion was decreased significantly in perfused livers from TR(-) compared with those from WT rats. In conclusion, expression and function of hepatic Bcrp are decreased significantly in TR(-) rats. The potential role of both Bcrp and Mrp2 should be considered when data generated in TR(-) rats are interpreted. TR(-) and EHBR rats in combination may be useful in differentiating the role of Mrp2 and Bcrp in drug/metabolite disposition.

Transcriptional modulation of BCRP gene to reverse multidrug resistance by toremifene in breast adenocarcinoma cells

Breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette half transporter, confers multidrug resistance (MDR) to a series of antitumor agents such as mitoxantrone, daunorubicin, SN-38, and topotecan, and often limits the efficacy of chemotherapy. Recent studies have indicated that a putative estrogen response element (ERE) is located in the promoter region of the BCRP gene. However, whether and how BCRP is regulated transcriptionally by toremifene (TOR) remains unknown. In the present study, two plasmid vectors have been designed to express the wild-type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a constitutive cytomegalovirus (CMV) promoter as control, respectively, which were transfected into estrogen-responsive MCF-7 and estrogen-independent MDA-MB-231 human breast adenocarcinoma cell lines. We showed that toremifene alone significantly downregulated BCRP mRNA and protein levels in estrogen receptor α (ERα)-positive MCF-7 cells in a dose-dependent manner, and the inhibitory effect was partially reversed by estrone (E(1)). Furthermore, gel shift assays demonstrated that specific binding of ERα to the ERE in the BCRP promoter is essential for transcriptional inhibition of BCRP by toremifene. Interestingly, toremifene alone increased the cellular accumulation of mitoxantrone in BCRP-transfected cells, suggesting that TOR indeed inhibits BCRP-mediated drug efflux and overcome drug resistance. To the best of our knowledge, this is the first report describing a direct effect of toremifene on BCRP. Our results thus indicate that toremifene by itself downregulates BCRP expression to reverse BCRP-mediated atypical multidrug resistance via a novel transcriptionally mechanism, which might be involved in TOR-ER complexes binding to the ERE of BCRP promoter to repress transcription of BCRP gene.

Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. In an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317, but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXRα knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile.

Regulation of BCRP (ABCG2) and P-glycoprotein (ABCB1) by cytokines in a model of the human blood-brain barrier.

Brain capillary endothelial cells form the blood-brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1beta, IL-6 and TNF-alpha. The strongest BCRP suppression at the protein level was observed after IL-1beta treatment. IL-1beta, IL-6 and TNF-alpha also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-alpha treatment. TNF-alpha also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.

Differential Effects of Chrysin on Nitrofurantoin Pharmacokinetics Mediated by Intestinal Breast Cancer Resistance Protein in Rats and Mice

The activities of breast cancer resistance protein (Bcrp/ABCG2) as well as P-glycoprotein (P-gp) and drug-metabolizing enzymes can be inhibited by several flavonoids or drugs in rats. However, the species, gender and regional differences of effects of flavonoids on Bcrp/ABCG2 in rats and mice remain unclear, although Bcrp, like P-gp, is also important in controlling drug absorption and disposition. We used chrysin as a model flavonoid because it possesses anti-inflammatory and antioxidative properties and is used as a dietary supplement. We examined the pharmacokinetics of nitrofurantoin, a specific Bcrp substrate, in rats and mice treated with chrysin. Bcrp mRNA levels were measured in liver, kidney, duodenum, jejunum and ileum in rats and mice. Plasma concentrations of nitrofurantoin were increased in rats, but not mice, treated with oral chrysin, compared with untreated controls. Intraperitoneal injection of chrysin into rats or mice had little effect on the elimination of nitrofurantoin, compared with untreated animals. These results suggest that chrysin-nitrofurantoin interactions occur in the small intestine in rats, but not in mice, possibly due to the higher levels of Bcrp expression in the small intestine in rats, compared with those in mice.

Decreased urinary secretion of belotecan in folic acid-induced acute renal failure rats due to down-regulation of Oat1 and Bcrp

The effects of folic acid-induced acute renal failure on the renal excretion of belotecan were investigated in rats after intravenous administration.Both glomeruli and renal tubules were seriously damaged by folic acid-induced acute renal failure. The renal excretion clearance, CLr, of belotecan was significantly decreased by folic acid-induced acute renal failure. Furthermore, glomerular filtration rate and secretion clearance of the drug were dramatically decreased by folic acid-induced acute renal failure.In vivo renal uptake of belotecan was inhibited by p-aminohippurate, whereas renal excretion was inhibited by GF120918, but not by verapamil and bromosulphalein. This indicates that Oat1/3 and Bcrp are involved in the renal uptake and urinary excretion of belotecan, respectively.Both mRNA and protein levels of Oat1, Oat3 and Bcrp were significantly decreased in folic acid-induced acute renal failure rats. Based on the finding that belotecan is a substrate of OAT1 but not of OAT3, the decrease in CLr of belotecan in folic acid-induced acute renal failure could, therefore, mainly be attributed to the down-regulation of Oat1 and Bcrp, in addition to the decrease in glomerular filtration rate.

Characterization of efflux proteins in human corneal epithelial cells

Abstract Purpose: Corneal epithelium is the main barrier for absorption of drugs into intraocular tissues after topical administration and part of this barrier may be formed by efflux proteins which translocate molecules from the cell interior to the extracellular space. The aim of this study was to characterize the gene expression and the activity of the efflux transporters in the cell culture model of immortalized human corneal epithelial cells (HCE cells), in primary cell line (HCEpiC), and in the human corneal epithelium. Methods: The mRNA levels of MDR1, MRP1‐MRP6, and BCRP were determined by the quantitative RT‐PCR. Immunohistochemistry was used to study protein expression and localization of efflux transporters. Functionality of these proteins was assessed with calcein‐AM efflux assay and by measuring the efflux of CDCF. Furthermore, bidirectional permeability of rhodamine 123 (Rh123) was studied. Results: The mRNA of MRP1 and MRP5 were detected in the human cornea and in both cell lines. These efflux proteins were found in the cell membranes of the human corneal epithelium. At mRNA level some efflux proteins were over‐expressed in the HCE and the primary cell lines. Increased calcein retention and decreased CDCF efflux in the presence of inhibitors suggested efflux protein activity in both primary and HCE cells. Likewise, directionality in Rh123 permeability was diminished in the presence of verapamil in HCE model. Conclusions: Functionality of the efflux proteins was demonstrated in the human corneal epithelial cells. MRP1 and MRP5 proteins may have important protecting role in corneal surface by transporting molecules out from the epithelial cells. It seems that the efflux activity in the HCE model differs from that of the corneal epithelium in vivo

Expression of breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation and at term before and after labor

Breast cancer resistance protein, BCRP, is a multidrug resistance protein that is highly expressed in the human placenta. In cancer tissues, this protein actively extrudes a wide variety of chemically and structurally unrelated chemotherapeutic drugs and other compounds. Studies in mice have shown that in the absence of BCRP activity in the placenta, there is a 2-fold increase in the uptake in BCRP substrates into fetus. This suggests that in the placenta, BCRP extrudes compounds that would otherwise cross the syncytiotrophoblast cells into fetal circulation. The purpose of this study was to examine the expression and localization of BCRP in the human placenta throughout gestation. Tissues from 6-13, 16-19, 24-29, 32-35, and 38-41 weeks of gestation were used. Real time RT-PCR analysis demonstrated that the mRNA levels of BCRP in the placenta do not change significantly as gestation progressed. However, Western blot analysis revealed that the protein levels increased towards the end of gestation. We demonstrated that BCRP is localized to the syncytiotrophoblast of the placenta and in some fetal blood vessels within the placenta. Tissues from the early stages of pregnancy (6-13 weeks) showed fewer BCRP positive blood vessels than term tissues (38-41 weeks).

OII-B-3Effect of maternal inflammation on the expression of ABCB1 and ABCG2 in placenta

BACKGROUND The placenta serves as a protective barrier for the fetus. The drug efflux transporters P-glycoprotein (Pgp/ABCB1) and BCRP (ABCG2) are highly expressed in placenta and diminish fetal exposure to xenobiotics. As endotoxin has been shown to suppress the expression of Pgp in many tissues, we examined its effect on Pgp and BCRP expression in placenta, and its impact on the transplacental passage of the Pgp substrate, 99mTc-MIBI. METHODS Pregnant rats were administered endotoxin (LPS, 0.5, 1.0 mg/kg i.p.) or saline (control) on G17. Rats received 99mTc-MIBI (20 MBq i.v.) 20h later, were sacrificed at 24h, and tissues collected. 99mTc-MIBI levels were measured in fetal and placental tissues. Expression of Pgp and BCRP was examined by RT-PCR and Western blots. RESULTS As compared to controls, placental levels of mdr1a/Pgp mRNA were significantly reduced by 57± 3 % and 88± 4 % and BCRP mRNA levels were significantly reduced by 55± 8 % and 99± 0.1 % of control values in rats administered 0.5 or 1.0 mg/kg LPS, respectively. Dose-dependent reductions in the protein expression of Pgp and BCRP were observed. Likewise, 3.5-fold higher levels of 99mTc-MIBI were seen in the fetuses of LPS treated rats (p < 0.05). CONCLUSIONS Endotoxin-induced inflammation imposed significant down-regulation of placental Pgp and BCRP and increased fetal drug accumulation. Hence, maternal diseases can significantly alter the expression and activity of ABC transporters and thereby may contribute to altered fetal drug exposure. Clinical Pharmacology & Therapeutics (2005) 79, P5–P5; doi: 10.1016/j.clpt.2005.12.016