Multidrug resistance protein 1 (MRP1, ABCC1) mediates resistance to mitoxantrone via glutathione-dependent drug efflux

Based upon several previous reports, no consistent relationship between multidrug resistance protein 1 (MRP1, ABCC1) expression and cellular sensitivity to mitoxantrone (MX) toxicity can be ascertained; thus, the role of MRP1 in MX resistance remains controversial. The present study, using paired parental, MRP1-poor, and transduced MRP1-overexpressing MCF7 cells, unequivocally demonstrates that MRP1 confers resistance to MX cytotoxicity and that resistance is associated with reduced cellular accumulation of MX. This MRP1-associated reduced accumulation of MX was partially reversed by treatment of cells with 50 microM MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]-an MRP inhibitor that increased MX accumulation in MRP1-expressing MCF7 cells but had no effect on MRP-poor MCF7 cells. Moreover, in vitro experiments using inside-out membrane vesicles show that MRP1 supports ATP-dependent, osmotically sensitive uptake of MX. Unlike ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein), MRP1-mediated MX transport is dependent upon the presence of glutathione or its S-methyl analog. In addition, MX stimulates transport of [3H]glutathione. Together, these data are consistent with the interpretation that MX efflux by MRP1 involves cotransport of MX and glutathione. The results suggest that MRP1-like the alternative MX transporters ABCG2 and ABCB1 (MDR1, P-glycoprotein)-can significantly influence tumor cell sensitivity to and pharmacological disposition of MX.     

Expression of lung resistance-related protein, LRP, and multidrug resistance-related protein, MRP1, in normal human lung cells in long-term cultures

Transport processes form part of the bodys defense mechanism, and they determine the intracellular levels of many endogenous and exogenous compounds. The multidrug resistance-related protein MRP1 and the lung resistance-related protein LRP are associated with drug resistance against chemotherapeutics; they protect cells against toxic compounds. There is much experimental evidence to suggest that both of these transporter proteins serve important physiological functions. The expression of LRP and MRP1 was studied in normal human bronchial epithelial cells (NHBEC) and peripheral lung cells (PLC) obtained from explant cultures from morphologically-normal human lung tissue taken from patients with lung cancer. LRP (mRNA and protein) was detected in the cells of the bronchi as well as the peripheral lung with low (a factor of 2.6) inter-individual variation in the first generation. No significant alterations were noted for LRP within three-to-four generations in the same patient. LRP expression was not substantially different between cultures from different topographic regions of the human lung. MRP1 protein and MRP1 mRNA could also be detected in all of the NHBEC and PLC cultures studied, but with substantially higher (a factor of 7.7) intra-individual variation in the first generation than for LRP. MRP expression was the same for bronchial cells and PLC when the material was obtained from both sites. The level of mRNA for MRP1 was, in general, less stable than that for LRP. In multigeneration explant cultures, the levels of LRP mRNA and protein and MRP1 protein did not fluctuate greatly, but the level of MRP1 mRNA dropped to about 25% of the reference value within four generations (after about 8–10 weeks of culture). In one case, NHBEC subpassages were followed over a period of 20 weeks. In this system MRP mRNA levels increased by more than threefold, while levels of MRP1 protein and LRP mRNA and protein were expressed at almost constant rates.     

Lipopolysaccharide Increases the Expression of Multidrug Resistance-Associated Protein 1 (MRP1) in RAW 264.7 Macrophages

Multidrug resistance-associated protein 1 (MRP-1) is a ubiquitously expressed member of the ATP-binding cassette transporter family. MRP-1 is one of the primary transporters of glutathione and glutathione conjugates. This protein also transports antiretroviral therapeutics, such as HIV-1 protease inhibitors (PI). We hypothesized that inflammatory mediators that activate macrophages would modify the expression and activity of MRP-1 in macrophages. Real-time PCR assays, western blots, and calcein efflux assays were used to show that exposure of macrophage cell line RAW 264.7 to lipopolysaccharide (LPS) increased expression of MRP-1 at the levels of mRNA, protein, and functional activity. Treatment of macrophages with LPS resulted in 2-fold increases of MRP-1 expression or functional activity. LPS-mediated increases in calcein efflux were repressed by the MRP-specific inhibitor MK-571. These results suggest that the effectiveness of HIV-1 PI therapy may be compromised by the presence of opportunistic infections.     

Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense

In tumor cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation which is attributed to the overexpression of certain ATP-binding cassette (ABC) transporter proteins. ABC proteins that confer drug resistance include (but are not limited to) P-glycoprotein (gene symbol ABCB1), the multidrug resistance protein 1 (MRP1, gene symbol ABCC1), MRP2 (gene symbol ABCC2), and the breast cancer resistance protein (BCRP, gene symbol ABCG2). In addition to their role in drug resistance, there is substantial evidence that these efflux pumps have overlapping functions in tissue defense. Collectively, these proteins are capable of transporting a vast and chemically diverse array of toxicants including bulky lipophilic cationic, anionic, and neutrally charged drugs and toxins as well as conjugated organic anions that encompass dietary and environmental carcinogens, pesticides, metals, metalloids, and lipid peroxidation products. P-glycoprotein, MRP1, MRP2, and BCRP/ABCG2 are expressed in tissues important for absorption (e.g., lung and gut) and metabolism and elimination (liver and kidney). In addition, these transporters have an important role in maintaining the barrier function of sanctuary site tissues (e.g., blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier or placenta). Thus, these ABC transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, excretion, and toxicity of xenobiotics. In this review, the role of these four ABC transporter proteins in protecting tissues from a variety of toxicants is discussed. Species variations in substrate specificity and tissue distribution of these transporters are also addressed since these properties have implications for in vivo models of toxicity used for drug discovery and development.     

Interaction of Oxazaphosphorines with Multidrug Resistance-Associated Protein 4 (MRP4)

Multidrug resistance-associated protein 4 (MRP4) is an organic anion efflux pump capable of transporting nucleoside, nucleotide analogs, and cyclic nucleotide. MRP4 could have an influence on the resistance and transport of the two oxazaphosphorines, cyclophosphamide (CP) and ifosfamide (IF). V/HepG2 (HepG2, hepatoma cells stably transfected with an empty vehicle plasmid) and MRP4/HepG2 (HepG2 cells stably expressing MRP4) were exposed to CP and IF in the absence or presence of various MRP4 inhibitors. HepG2 and HEK293 human kidney cells were also used to investigate the inducing potency of oxazaphosphorines on the MRP4 expression. In this study, insertion of MRP4 gene in HepG2 cells was found to confer significant resistance to CP and IF in the 48-h drug-exposure assays. In the presence of various MRP4 inhibitors, the resistance to CP and IF was then partially reversed. These indicate that CP and IF are highly possible substrates of MRP4. In addition, CP and clofibrate (CFB), a reported MRP4 inducer, in vivo significantly increased the MRP4 expression at both protein level and mRNA level in HEK293 cells at higher concentrations, while IF significantly decreased the MRP4 expression at mRNA level at lower concentration and had no effect at higher concentrations. However, all tested compounds (CP, IF, and CFB) did not change the MRP4 protein expression in HepG2 cells. CP and CFB are cell-specific and concentration-dependent MRP4 inducers. The finding may have implications in the CP- or IF-based chemotherapy.     

Multidrug resistance-associated protein 2 (MRP2) enhances 4-hydroxynonenal-induced toxicity in Madin-Darby canine kidney II cells

4-Hydroxy-trans-2,3-nonenal (HNE) is a toxic end product of lipid peroxidation. This multifunctional aldehyde reacts with proteins, phospholipids, and nucleic acids, consequently activating/inactivating enzymes, affecting signal transduction and gene expression. HNE is mainly detoxified by glutathione (GSH) conjugation. In our previous report, we showed that GSH conjugates of 4-hydroxynonenal (HNE-SG) are substrates of multidrug resistance-associated protein 2 (MRP2). MRP2 has been shown to export HNE-SG conjugates into the extracellular space. In the present study, the role of MRP2 in the detoxification of HNE was studied using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2. MRP2 reduced the intracellular accumulation of HNE-SG conjugate but unexpectedly increased the susceptibility of cells to HNE. The viability of cells was reduced to approximately 70% in the presence of 62.5 microM HNE in MDCK II cells expressing MRP2, whereas MDCK II cells remained unaffected. MRP2 accelerated the elimination of intracellular GSH via a conjugation reaction with HNE (half-life of GSH was 30.1 and 12.2 min for MDCK II cells and MDCK II cells expressing MRP2, respectively). Moreover, the consumption of GSH was unlimited in MDCK II cells expressing MRP2, finally resulting in necrosis. These results indicate that MRP2 has an adverse effect during the detoxification of HNE in MDCK II cells and suggest that expression of MRP2 may enhance the damage caused by oxidative stress.     

Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugs and signalling molecules

Multidrug resistance protein (MRP) 4 is a member of the MRP/ABCC subfamily of ATP-binding cassette transporters, which are capable of pumping a wide variety of endogenous and xenobiotic organic anionic compounds out of the cell. In addition to its role in the body distribution and renal excretion of a wide variety of antiviral, cytostatic, antibiotic and cardiovascular drugs, MRP4/ABCC4 has the remarkable ability to transport molecules involved in cellular signalling. These molecules include cyclic nucleotides, eicosanoids, urate and conjugated steroids. The unique structure, regulation and dual localisation of MRP4 in polarised cells could be connected with a key function in cellular protection and extracellular signalling pathways. This review focuses on recent insights into the versatile transport function of MRP4 and its potential as a new therapeutic target to modulate various pathophysiological signalling processes.     

Inhibition of Multidrug Resistance-Associated Protein (MRP) Functional Activity with Pluronic Block Copolymers

Purpose. Using monolayers of human pancreatic adenocarcinoma cells (Panc-1) that express multidrug resistance-associated protein (MRP), the present work investigates the effects of Pluronic block copolymers on the functional activity of MRP. Methods. The studies examined the accumulation and efflux of the MRP selective probe fluorescein (FLU) in Panc-1 cell monolayers with and without Pluronic P85 (P85), Pluronic L81 (L81) and Pluronic F108 (F108). Results. Treatment of Panc-1 cells with P85 resulted in concentration-dependent increases in FLU accumulation and elimination of FLU sequestration in vesicular compartments in these cells. The effects of P85 were selective for FLU in the Panc-1 cell monolayers. Inhibition of MRP-mediated transport was dependent on the composition of Pluronic block copolymer: the more hydrophobic copolymer had the greater effect on FLU uptake in Panc-1 monolayers (L81 > P85 > F108). Conclusions. This paper demonstrates for the first time that Pluronic block copolymers inhibit multidrug resistance-associated protein (MRP). The similarities in the effects of Pluronic block copolymers on MRP and P-glycoprotein drug efflux systems suggest that a single unifying mechanism may explain the inhibition observed.     

Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.     

Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[(32)P]-triphosphate ([(32)P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [gamma-(32)P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [alpha-(32)P]azidoADP. [alpha-(32)P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.     

Inhibition of the multidrug resistance protein 1 (MRP1) by peptidomimetic glutathione-conjugate analogs

Inhibition of multidrug resistance protein 1 (MRP1) mediated cytostatic drug efflux might be useful in the treatment of drug resistant tumors. Because the glutathione (GSH) conjugate of ethacrynic acid (EA), GS-EA, is a good substrate of MRP1, GS-EA derivatives are expected to be good inhibitors of MRP1. To study structure-activity relationships of MRP1 inhibition, a series of novel GS-EA analogs was synthesized in which peptide bonds of the GSH backbone were replaced by isosteric groups [Bioorg Med Chem 10:195-205, 2002]. Several of these compounds were effective inhibitors of MRP1-mediated [(3)H]GS-EA and [(3)H]E(2)17betaG transport, as studied in membrane vesicles prepared from MRP1-overproducing Sf9 cells. The modifications of the peptide backbone have distinct implications for recognition by MRP1: the gamma-glutamyl-cysteine peptide bond is important for binding, whereas the cysteinyl-glycine amide does not seem essential. When the gamma-glutamyl-cysteine peptide bond (C-CO-N) is replaced by a urethane isostere (O-CO-N), an effective competitive MRP1-inhibitor (K(i) = 11 microM) is obtained. After esterification of this compound to improve its cellular uptake, it inhibited MRP1-mediated efflux of calcein from 2008 ovarian carcinoma cells overexpressing MRP1. This compound also partially reversed the resistance of these cells to methotrexate. Because the urethane isostere is stable toward gamma-glutamyl transpeptidase-mediated breakdown, it is an interesting lead-compound for the development of in vivo active MRP1 inhibitors.     

Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (−)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2′-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (−)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (−)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.     

Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters

The 190 kDa multidrug resistance protein 1 (MRP1/ABCC1) is a founding member of a subfamily of the ATP binding cassette (ABC) superfamily of transport proteins and was originally identified on the basis of its elevated expression in multidrug resistant lung cancer cells. In addition to its ability to confer resistance in tumour cells, MRP1 is ubiquitously expressed in normal tissues and is a primary active transporter of GSH, glucuronate and sulfate conjugated and unconjugated organic anions of toxicological relevance. Substrates include lipid peroxidation products, herbicides, tobacco specific nitrosamines, mycotoxins, heavy metals, and natural product and antifolate anti-cancer agents. MRP1 also transports unmodified xenobiotics but often requires GSH to do so. Active efflux is generally an important aspect of cellular detoxification since it prevents the accumulation of conjugated and unconjugated compounds that have the potential to be directly toxic. The related transporters MRP2 and MRP3 have overlapping substrate specificities with MRP1 but different tissue distributions, and evidence that they also have chemoprotective functions are discussed. Finally, MRP homologues have been described in other species including yeast and nematodes. Those isolated from the vascular plant Arabidopsis thaliana (AtMRPs) decrease the cytoplasmic concentration of conjugated toxins through sequestration in vacuoles and are implicated in providing herbicide resistance to plants.     

Multidrug resistance-associated protein 2 (MRP2) affects hepatobiliary elimination but not the intestinal disposition of tenofovir disoproxil fumarate and its metabolites

The role of multidrug resistance-associated protein 2 (MRP2) on the intestinal disposition and hepatobiliary elimination of tenofovir disoproxil fumarate (DF) and its metabolites [tenofovir (mono)ester and tenofovir] was studied in the Caco-2 system, Ussing chambers and rat in-situ efflux experiments. In the Caco-2 model and Ussing chambers, no statistically significant differences in transport could be observed when the MRP inhibitor probenecid was included. In Ussing chambers, transport was also similar when using intestinal tissue from MRP2-deficient rats. After intravenous administration of tenofovir DF, the excretion of tenofovir [(mono)ester] in bile was significantly decreased in MRP2-deficient rats and in rats treated with probenecid. The area under the blood concentration-time curve was increased in MRP2-deficient rats [1.0+/-0.1 and 0.36+/-0.03 microM.min-1 for tenofovir and tenofovir (mono)ester, respectively] and rats treated with probenecid (1.42+/-0.04 and 0.36+/-0.02 microM.min-1) compared with control rats (0.64+/-0.05 and 0.15+/-0.06 microM.min-1). The appearance of tenofovir [(mono)ester] in intestinal perfusate was similar in control rats upon co-administering probenecid or when using MRP2-deficient rats. In conclusion, MRP2 appeared to have no modulatory effect on the intestinal disposition of tenofovir and tenofovir (mono)ester. However, inhibition (probenecid) or the total absence of MRP2 (MRP2-deficient rats) significantly reduced hepatobiliary elimination, which was accompanied by increased systemic exposure.     

Comparison of furosemide and vinblastine secretion from cell lines overexpressing multidrug resistance protein (P-glycoprotein) and multidrug resistance-associated proteins (MRP1 and MRP2)

Recent studies in our laboratory have shown that the loop diuretic, furosemide, is actively secreted by Caco-2 cells and rat jejunal tissue. This active secretion could be the result of efflux transporters such as P-gp, MRP1 or MRP2 (cMOAT). To determine if any of these transporters is responsible for the secretion of furosemide, we compared directional permeability in the wild-type cell lines, MDCK strains I and II, and LLC-PK1, vs. cell lines that overexpress a single transporter, in both the presence and absence of various inhibitors, for furosemide as compared to vinblastine. Sulfinpyrazone significantly inhibited the transport of vinblastine in MRP2 expressing cells, but not the wild-type controls. Vinblastine could not be confirmed as a substrate of MRP1. We were also unable to demonstrate that any particular transporter affected furosemide in excess of the background effects of endogenous transporters in the parental cell lines. Furosemide secretion from these kidney-derived cell lines is probably not the primary result of any of the well characterized efflux transporters (P-gp, MRP1 or MRP2), although they may still play a role in the observed Caco-2 secretion. This equivocal result acknowledges the difficulty in trying to determine the effect of a single protein in a complicated expression system.     

Multidrug resistance-associated protein 2 (MRP2) affects hepatobiliary elimination but not the intestinal disposition of tenofovir disoproxil fumarate and its metabolites

The role of multidrug resistance-associated protein 2 (MRP2) on the intestinal disposition and hepatobiliary elimination of tenofovir disoproxil fumarate (DF) and its metabolites [tenofovir (mono)ester and tenofovir] was studied in the Caco-2 system, Ussing chambers and rat in-situ efflux experiments. In the Caco-2 model and Ussing chambers, no statistically significant differences in transport could be observed when the MRP inhibitor probenecid was included. In Ussing chambers, transport was also similar when using intestinal tissue from MRP2-deficient rats. After intravenous administration of tenofovir DF, the excretion of tenofovir [(mono)ester] in bile was significantly decreased in MRP2-deficient rats and in rats treated with probenecid. The area under the blood concentration–time curve was increased in MRP2-deficient rats [1.0?±?0.1 and 0.36?±?0.03?µM.min^?1 for tenofovir and tenofovir (mono)ester, respectively] and rats treated with probenecid (1.42?±?0.04 and 0.36?±?0.02?µM.min^?1) compared with control rats (0.64?±?0.05 and 0.15?±?0.06?µM.min^?1). The appearance of tenofovir [(mono)ester] in intestinal perfusate was similar in control rats upon co-administering probenecid or when using MRP2-deficient rats. In conclusion, MRP2 appeared to have no modulatory effect on the intestinal disposition of tenofovir and tenofovir (mono)ester. However, inhibition (probenecid) or the total absence of MRP2 (MRP2-deficient rats) significantly reduced hepatobiliary elimination, which was accompanied by increased systemic exposure.     

Monomethylarsenic diglutathione transport by the human multidrug resistance protein 1 (MRP1/ABCC1)

The ATP-binding cassette (ABC) transporter protein multidrug resistance protein 1 (MRP1; ABCC1) plays an important role in the cellular efflux of the high-priority environmental carcinogen arsenic as a triglutathione conjugate [As(GS)(3)]. Most mammalian cells can methylate arsenic to monomethylarsonous acid (MMA(III)), monomethylarsonic acid (MMA(V)), dimethylarsinous acid (DMA(III)), and dimethylarsinic acid (DMA(V)). The trivalent forms MMA(III) and DMA(III) are more reactive and toxic than their inorganic precursors, arsenite (As(III)) and arsenate (As(V)). The ability of MRP1 to transport methylated arsenicals is unknown and was the focus of the current study. HeLa cells expressing MRP1 (HeLa-MRP1) were found to confer a 2.6-fold higher level of resistance to MMA(III) than empty vector control (HeLa-vector) cells, and this resistance was dependent on GSH. In contrast, MRP1 did not confer resistance to DMA(III), MMA(V), or DMA(V). HeLa-MRP1 cells accumulated 4.5-fold less MMA(III) than HeLa-vector cells. Experiments using MRP1-enriched membrane vesicles showed that transport of MMA(III) was GSH-dependent but not supported by the nonreducing GSH analog, ophthalmic acid, suggesting that MMA(III)(GS)(2) was the transported form. MMA(III)(GS)(2) was a high-affinity, high-capacity substrate for MRP1 with apparent K(m) and V(max) values of 11 μM and 11 nmol mg(-1)min(-1), respectively. MMA(III)(GS)(2) transport was osmotically sensitive and inhibited by several MRP1 substrates, including 17β-estradiol 17-(β-D-glucuronide) (E(2)17βG). MMA(III)(GS)(2) competitively inhibited the transport of E(2)17βG with a K(i) value of 16 μM, indicating that these two substrates have overlapping binding sites. These results suggest that MRP1 is an important cellular protective pathway for the highly toxic MMA(III) and have implications for environmental and clinical exposure to arsenic.     

Structure and function of the MRP2 (ABCC2) protein and its role in drug disposition

The multi-drug resistance protein 2 (MRP2; ABCC2) is an ATP-binding cassette transporter playing an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, glucuronate and sulfate, which are collectively known as phase II products of biotransformation. In addition, MRP2 can also transport uncharged compounds in cotransport with glutathione, and thus can modulate the pharmacokinetics of many drugs. The other way around, its expression and activity are also altered by certain drugs and disease states. Unlike other members of the MRP/ABCC family, MRP2 is specifically expressed on the apical membrane domain of polarised cells as hepatocytes, renal proximal tubular cells, enterocytes and syncytiotrophoblasts of the placenta. Several naturally occurring mutations leading to the absence of functional MRP2 protein from the apical membrane have been described causing the human Dubin-Johnson syndrome associated with conjugated hyperbilirubinaemia. Experimental mutation studies have revealed critical amino acids for substrate binding in the MRP2 molecule. This review is, therefore, focused on the structure and function of MRP2, the substrates transported and the clinical relevance of MRP2.     

Verapamil-stimulated glutathione transport by the multidrug resistance-associated protein (MRP1) in leukaemia cells.

Multidrug resistance mediated by the multidrug resistance-associated protein MRP1 is associated with decreased drug accumulation, which is in turn dependent on cellular glutathione. We have reported that verapamil, an inhibitor of drug transport, caused a decrease in cellular glutathione in CCRF-CEM/E1000 MRP1-overexpressing leukaemia cells (Biochem Pharmacol 55;1283--9, 1998). We now demonstrate that other inhibitors of MRP1-mediated drug transport (e.g. MK571, indomethacin, genistein, and nifedipine) deplete cellular glutathione in these leukaemia cells (>30% decrease; P < 0.01) while having no effect on the parental CCRF-CEM cells. However, treatment with etoposide or vincristine (at similar molar concentrations) caused a 20% decrease in glutathione. Verapamil-stimulated glutathione transport correlated with MRP1 expression in a series of drug-resistant cells, and glutathione was quantitatively recovered in the extracellular media. Further, verapamil-stimulated glutathione transport was rapid (50% decrease in 10 min), dose-dependent, and inhibited by vanadate, an inhibitor of ATPase activity, but not by sulphobromophthalein (BSP) or methionine, inhibitors of hepatic glutathione transporters. Incubation of CCRF-CEM/E1000 cells in 25 mM glutathione not only showed that verapamil-mediated efflux occurred against the concentration gradient, but also demonstrated the MRP1-mediated uptake of glutathione (P < 0.01 compared to the parental CCRF-CEM cells), which was not inhibited by vanadate. These results demonstrate that while MRP1 transports glutathione in the presence of inhibitors of drug transport, there is no convincing evidence for co-transport of glutathione with drug. They further demonstrate that MRP1 mediates the facilitated transport of glutathione into the MRP1-overexpressing CEM/E1000 cells, suggesting that MRP1 may play a major role in cellular glutathione homeostasis.