Role of multidrug resistance protein 1 (MRP1) and glutathione S-transferase A1-1 in alkylating agent resistance. Kinetics of glutathione conjugate formation and efflux govern differential cellular sensitivity to chlorambucil versus melphalan toxicity

We investigated the role of phase II (conjugation) and phase III (efflux) detoxification of the anticancer drugs melphalan (MLP) and chlorambucil (CHB). Although both drugs are substrates of Alpha-class glutathione S-transferases (GST) and the monoglutathionyl conjugates formed in these enzymatic reactions are transported by MRP1, we found that GSTA1-1 and MRP1 acted in synergy to confer resistance to CHB but not to MLP (Morrow, C. S., Smitherman, P. K., Diah, S. K., Schneider, E., and Townsend, A. J. (1998) J. Biol. Chem. 273, 20114-20120). To explain this selectivity of MRP1/GST-mediated resistance, we report results of side-by-side experiments comparing the kinetics of MLP- versus CHB-glutathione conjugate: formation, product inhibition of GSTA1-1 catalysis, and transport by MRP1. The monoglutathionyl conjugate of CHB, CHB-SG, is a very strong competitive inhibitor of GSTA1-1 (K(i) 0.14 microM) that is >30-fold more potent than that of the corresponding conjugate of MLP, MLP-SG (K(i) 4.7 microM). The efficiency of GSTA1-1-mediated monoglutathionyl conjugate formation is more than 4-fold higher for CHB than MLP. Lastly, both CHB-SG and MLP-SG are efficiently transported by MRP1 with similar V(max) although the K(m) for CHB-SG (0.37 microm) is significantly lower than for MLP-SG (1.1 microM). These results indicate that MRP1 is required for GSTA1-1-mediated resistance to CHB in order to relieve potent product inhibition of the enzyme by intracellular CHB-SG formed. The kinetic properties of MRP1 are well suited to eliminate CHB-SG at pharmacologically relevant concentrations. For MLP detoxification, where product inhibition of GSTA1-1 is less important, GSTA1-1 does not confer resistance because of the relatively poorer catalytic efficiency of MLP-SG formation. Similar analyses can be useful for predicting the pharmacological and toxicological consequences of MRP and GST expression on cellular sensitivity to various other electrophilic xenobiotics.     

Nitric oxide storage and transport in cells are mediated by glutathione S-transferase P1-1 and multidrug resistance protein 1 via dinitrosyl iron complexes

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron release. We showed that NO-mediated iron efflux from cells required glutathione (GSH) (Watts, R. N., and Richardson, D. R. (2001) J. Biol. Chem. 276, 4724-4732) and that the GSH-conjugate transporter, multidrug resistance-associated protein 1 (MRP1), mediates this release potentially as a dinitrosyl-dithiol iron complex (DNIC; Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675). Recently, glutathione S-transferase P1-1 (GST P1-1) was shown to bind DNICs as dinitrosyl-diglutathionyl iron complexes. Considering this and that GSTs and MRP1 form an integrated detoxification unit with chemotherapeutics, we assessed whether these proteins coordinately regulate storage and transport of DNICs as long lived NO intermediates. Cells transfected with GSTP1 (but not GSTA1 or GSTM1) significantly decreased NO-mediated 59Fe release from cells. This NO-mediated 59Fe efflux and the effect of GST P1-1 on preventing this were observed with NO-generating agents and also in cells transfected with inducible nitric oxide synthase. Notably, 59Fe accumulated in cells within GST P1-1-containing fractions, indicating an alteration in intracellular 59Fe distribution. Furthermore, electron paramagnetic resonance studies showed that MCF7-VP cells transfected with GSTP1 contain significantly greater levels of a unique DNIC signal. These investigations indicate that GST P1-1 acts to sequester NO as DNICs, reducing their transport out of the cell by MRP1. Cell proliferation studies demonstrated the importance of the combined effect of GST P1-1 and MRP1 in protecting cells from the cytotoxic effects of NO. Thus, the DNIC storage function of GST P1-1 and ability of MRP1 to efflux DNICs are vital in protection against NO cytotoxicity.     

Arsenic transport by the human multidrug resistance protein 1 (MRP1/ABCC1). Evidence that a tri-glutathione conjugate is required

Inorganic arsenic is an established human carcinogen, but its metabolism is incompletely defined. The ATP binding cassette protein, multidrug resistance protein (MRP1/ABCC1), transports conjugated organic anions (e.g. leukotriene C(4)) and also co-transports certain unmodified xenobiotics (e.g. vincristine) with glutathione (GSH). MRP1 also confers resistance to arsenic in association with GSH; however, the mechanism and the species of arsenic transported are unknown. Using membrane vesicles prepared from the MRP1-overexpressing lung cancer cell line, H69AR, we found that MRP1 transports arsenite (As(III)) only in the presence of GSH but does not transport arsenate (As(V)) (with or without GSH). The non-reducing GSH analogs L-gamma-glutamyl-L-alpha-aminobutyryl glycine and S-methyl GSH did not support As(III) transport, indicating that the free thiol group of GSH is required. GSH-dependent transport of As(III) was 2-fold higher at pH 6.5-7 than at a more basic pH, consistent with the formation and transport of the acid-stable arsenic triglutathione (As(GS)(3)). Immunoblot analysis of H69AR vesicles revealed the unexpected membrane association of GSH S-transferase P1-1 (GSTP1-1). Membrane vesicles from an MRP1-transfected HeLa cell line lacking membrane-associated GSTP1-1 did not transport As(III) even in the presence of GSH but did transport synthetic As(GS)(3). The addition of exogenous GSTP1-1 to HeLa-MRP1 vesicles resulted in GSH-dependent As(III) transport. The apparent K(m) of As(GS)(3) for MRP1 was 0.32 microM, suggesting a remarkably high relative affinity. As(GS)(3) transport by MRP1 was osmotically sensitive and was inhibited by several conjugated organic anions (MRP1 substrates) as well as the metalloid antimonite (K(i) 2.8 microM). As(GS)(3) transport experiments using MRP1 mutants with substrate specificities differing from wild-type MRP1 suggested a commonality in the substrate binding pockets of As(GS)(3) and leukotriene C(4). Finally, human MRP2 also transported As(GS)(3). In conclusion, MRP1 transports inorganic arsenic as a tri-GSH conjugate, and GSTP1-1 may have a synergistic role in this process.     

Multidrug resistance protein 4 (MRP4/ABCC4) mediates efflux of bimane-glutathione

Multidrug resistance proteins (MRPs) are ATP-dependent export pumps that mediate the export of organic anions. ABCC1 (MRP1), ABCC2 (MRP2) and ABCC3 (MRP3) are all able to facilitate the efflux of anionic conjugates including glutathione (GSH), glucuronide and sulfate conjugates of xenobiotics and endogenous molecules. Earlier studies showed that ABCC4 functions as an ATP-driven export pump for cyclic AMP and cyclic GMP, as well as estradiol-17-beta-D-glucuronide. However, it was unclear if other conjugated metabolites can be transported by ABCC4. Hence in this study, a fluorescent substrate, bimane-glutathione (bimane-GS) was used to further examine the transport activity of ABCC4. Using cells stably overexpressing ABCC4, this study shows that ABCC4 can facilitate the efflux of the glutathione conjugate, bimane-glutathione. Bimane-glutathione efflux increased with time and >85% of the conjugate was exported after 15min. This transport was abolished in the presence of 2.5microM carbonylcyanide m-chlorophenylhydrasone (CCCP), an uncoupler of oxidative phosphorylation. Inhibition was also observed with known inhibitors of MRP transporters including benzbromarone, verapamil and indomethacin. In addition, 100microM methotrexate, an ABCC4 substrate or 100microM 6-thioguanine (6-TG), a compound whose monophosphate metabolite is an ABCC4 substrate, reduced efflux by >40%. A concentration-dependent inhibition of bimane-glutathione efflux was observed with 1-chloro-2,4-dinitrobenzene (CDNB) which is metabolized intracellularly to the glutathione conjugate, 2,4-dinitrophenyl-glutathione (DNP-GS). The determination that ABCC4 can mediate the transport of glucuronide and glutathione conjugates indicates that ABCC4 may play a role in the cellular extrusion of Phase II detoxification metabolites.     

Export pumps for glutathione S-conjugates

The release of glutathione S-conjugates from cells is an ATP-dependent process mediated by integral membrane glycoproteins belonging to the recently discovered multidrug-resistance protein (MRP) family. Many lipophilic compounds conjugated with glutathione, glucuronate, or sulfate are substrates for export pumps of the MRP family. In humans six MRP isoforms encoded by different genes have been cloned. Orthologs of MRP have been identified in many species including yeast, plants, and nematodes. Human MRP1 and MRP2 are currently best characterized with respect to substrate specificity by measurements of ATP-dependent transport into inside-out membrane vesicles. High-affinity substrates include the glutathione S-conjugate leukotriene C4, S-(2,4dinitrophenyl)glutathione, bilirubin glucuronosides, and 17beta-glucuronosyl estradiol. In addition, glutathione disulfide is transported by MRP1 and MRP2. Reduced glutathione may be released from cells in a process directly or indirectly mediated by members of the MRP family. Proteins of the MRP family are indispensable for transport of glutathione S-conjugates and glutathione disulfide into the extracellular space and play, therefore, a decisive role in detoxification and defense against oxidative stress.     

Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione,glucuronate or sulfate

Many endogenous or xenobiotic lipophilic substances are eliminated from the cells by the sequence of oxidation, conjugation to an anionic group (glutathione, glucuronate or sulfate) and transport across the plasma membrane into the extracellular space. The latter step is mediated by integral membrane glycoproteins belonging to the superfamily of ATP-Binding Cassette (ABC) transporters. A subfamily, referred as ABCC, includes the famous/infamous cystic fibrosis transmembrane regulator (CFTR), the sulfonylurea receptors (SUR 1 and 2), and the multidrug resistance-associated proteins (MRPs). The name of the MRPs refers to their potential role in clinical multidrug resistance, a phenomenon that hinders the effective chemotherapy of tumors. The MRPs that have been functionally characterized so far share the property of ATP-dependent export pumps for conjugates with glutathione (GSH), glucuronate or sulfate. MRP1 and MRP2 are also mediating the cotransport of unconjugated amphiphilic compounds together with free GSH. MRP3 preferentially transports glucuronides but not glutathione S-conjugates or free GSH. MRP1 and MRP2 also contribute to the control of the intracellular glutathione disulfide (GSSG) level. Although these proteins are low affinity GSSG transporters, they can play essential role in response to oxidative stress when the activity of GSSG reductase becomes rate limiting. The human MRP4, MRP5 and MRP6 have only partially been characterized. However, it has been revealed that MRP4 can function as an efflux pump for cyclic nucleotides and nucleoside analogues, used as anti-HIV drugs. MRP5 also transports GSH conjugates, nucleoside analogues, and possibly heavy metal complexes. Transport of glutathione S-conjugates mediated by MRP6, the mutation of which causes pseudoxantoma elasticum, has recently been shown. In summary, numerous members of the multidrug resistance-associated protein family serve as export pumps that prevent the accumulation of anionic conjugates and GSSG in the cytoplasm, and play, therefore, an essential role in detoxification and defense against oxidative stress.     

Enhanced glutathione depletion, protein adduct formation, and cytotoxicity following exposure to 4-hydroxy-2-nonenal (HNE) in cells expressing human multidrug resistance protein-1 (MRP1) together with human glutathione S-transferase-M1 (GSTM1)

4-Hydroxy-2-nonenal (HNE) is one of the most reactive products of lipid peroxidation and has both cytotoxic and genotoxic effects in cells. Several enzymatic pathways have been reported to detoxify HNE, including conjugation by glutathione-S-transferases (GSTs). Removal of the resulting HNE-glutathione conjugate (HNE-SG) by an efflux transporter may be required for complete detoxification. We investigated the effect of expression of GSTM1 and/or the ABC efflux transporter protein, multidrug-resistance protein-1 (MRP1), on HNE-induced cellular toxicity. Stably transfected MCF7 cell lines were used to examine the effect of GSTM1 and/or MRP1 expression on HNE-induced cytotoxicity, GSH depletion, and HNE-protein adduct formation. Co-expression in the MCF7 cell line of GSTM1 with MRP1 resulted in a 2.3-fold sensitization to HNE cytotoxicity (0.44-fold IC(50) value relative to control) rather than the expected protection. Expression of either GSTM1 or MRP1 alone also resulted in slight sensitization to HNE cytotoxicity (0.79-fold and 0.71-fold decreases in IC(50) values, respectively). Co-expression of GSTM1 and MRP1 strongly enhanced the formation of HNE-protein adducts relative to the non-expressing control cell line, whereas expression of either MRP1 alone or GSTM1 alone yielded similarly low levels of HNE-protein adducts to that of the control cell line. Glutathione (GSH) levels were reduced by 10-20% in either the control cell line or the MCF7/GSTM1 cell line with the same HNE exposure for 60min. However, HNE induced >80% depletion of GSH in cells expressing MRP1 alone. Co-expression of both MRP1 and GSTM1 caused slightly greater GSH depletion, consistent with the greater protein adduct formation and cytotoxicity in this cell line. Since expression of GSTM1 or MRP1 alone did not strongly sensitize cells to HNE, or result in greater HNE-protein adducts than in the control cell line, these results indicate that MRP1 and GSTM1 collaborate to enhance HNE-protein adduct formation and HNE cytotoxicity, facilitated by GSH depletion mediated by both MRP1 and GSTM1.     

Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.     

Role of the Multidrug Resistance Protein 1 in protection from heavy metal oxyanions: investigations in vitro and in MRP1-deficient mice

The Multidrug Resistance Protein 1 (MRP1) is a membrane pump that mediates the efflux of a wide variety of xenobiotics, including arsenical and antimonial compounds, as demonstrated by the study of MRP1-transfected cell lines. We have previously shown that mrp1(-/-) cells are hypersensitive to sodium arsenite, sodium arsenate, and antimony potassium tartrate. We now report that the retroviral vector-mediated overexpression of MRP1 and of the two subunits of gamma-GCS (heavy and light) resulted in higher intracellular glutathione levels and in a greater level of resistance to sodium arsenite and antimony potassium tartrate, compared to the overexpression of MRP1 and gamma-GCS heavy alone. These observations further demonstrate that glutathione is an important component of MRP1-mediated cellular resistance to arsenite and antimony. However, the constitutive expression of MRP1 did not protect mice from the lethality of sodium arsenite and antimony potassium tartrate nor reduced the tissue accumulation of arsenic in mice injected i.p. with sodium arsenite. It is conceivable that, in vivo, other pump(s) effectively vicariate for MRP1-mediated transport of heavy metal oxyanions.     

Glutathione stimulates sulfated estrogen transport by multidrug resistance protein 1

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette (ABC) transporter that transports a range of hydrophobic xenobiotics, as well as relatively hydrophilic organic anion conjugates. The protein is present at high levels in testicular Leydig and Sertoli cells. Studies with knockout mice suggest that MRP1 may protect germ cells from exposure to some cytotoxic xenobiotics, but potential endobiotic substrates in this organ have not been identified. Previously, we have shown certain D-ring, but not A-ring, estrogen glucuronides can act as competitive inhibitors of MRP1 mediated transport, suggesting that they are potential substrates for the protein. In the case of 17 beta-estradiol-17 beta-d-glucuronide, this has been confirmed by direct transport studies. The Leydig cell is the major site of estrogen conjugation in the testis. However, the principal products of conjugation are A-ring estrogen sulfates, which are then effluxed from the cell by an unknown transporter. To determine whether MRP1/mrp1 could fulfill this function, we used membrane vesicles from MRP1-transfected HeLa cells to assess this possibility. We found that estradiol and estrone 3-sulfate alone were poor competitors of MRP1-mediated transport of the cysteinyl leukotriene, leukotriene C(4). However, in the presence of reduced glutathione (GSH), their inhibitory potency was markedly increased. Direct transport studies using [(3)H]estrone 3-sulfate confirmed that the conjugated estrogen could be efficiently transported (K(m) = 0.73 microm, V(max) = 440 pmol mg(-)1 protein min(-)1), but only in the presence of either GSH or the nonreducing alkyl derivative, S-methyl GSH. In contrast to previous studies using vincristine as a substrate, we detected no reciprocal increase in MRP1-mediated GSH transport. These results provide the first example of GSH-stimulated, MRP1-mediated transport of a potential endogenous substrate and expand the range of MRP1 substrates whose transport is stimulated by GSH to include certain hydrophilic conjugated endobiotics, in addition to previously identified hydrophobic xenobiotics.     

Excretion of glutathione conjugates by primary cultured rat hepatocytes

Conjugation of xenobiotics with glutathione occurs commonly within the liver, and these glutathione conjugates are then preferentially excreted into bile. We have characterized this excretory process using primary cultured hepatocytes (24 h). 1-Chloro-2,4-dinitrobenzene rapidly entered the cells and formed a glutathione conjugate, S-(dinitrophenyl)glutathione, irrespective of the temperature of incubation. In contrast, the efflux of the glutathione conjugate was essentially absent in the cold but recovered rapidly upon rewarming of the cells. Therefore, initial rates of efflux of the conjugate at 37 degrees C were measured from cells preloaded biosynthetically at 10 degrees C. Efflux was a saturable process with respect to intracellular S-(dinitrophenyl)glutathione with an apparent Km of 0.58 +/- 0.12 mM and Vmax of 0.15 +/- 0.05 nmol/min/mg of protein. The excretion of S-(dinitrophenyl)glutathione had an energy of activation of 15.3 kcal/mol. The glutathione conjugate of p-nitrobenzylchloride when formed within the hepatocytes acted as a competitive inhibitor of S-(dinitrophenyl)glutathione efflux. Cultured hepatocytes, therefore, appeared to have a specific transport process for the excretion of glutathione conjugates. The addition of S-(dinitrophenyl)glutathione, but not GSH, GSSG, or methionine, to the medium caused a decrease in the rate of efflux of radiolabeled S-(dinitrophenyl)glutathione. The hepatocytes were able, however, to excrete the glutathione conjugate against an excess of extracellular S-(dinitrophenyl)glutathione. This observation suggested that extracellular S-(dinitrophenyl)glutathione, although capable of binding to the carrier, entered the hepatocytes quite slowly relative to rates of efflux. This carrier may function in a manner that would minimize the reuptake by hepatocytes of conjugates that have been excreted into the bile.     

Human MDR1 and MRP1 Recognize Berberine as Their Transport Substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.     

Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor gamma (PPAR gamma) ligand, 15-deoxy-delta 12,14prostaglandin J2 (15-d-PGJ2)

15-Deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), a terminal metabolite of the J-series cyclopentenone prostaglandins, influences a variety of cellular processes including gene expression, differentiation, growth, and apoptosis. As a ligand of peroxisomal proliferator-activated receptor gamma (PPAR gamma), 15-d-PGJ(2) can transactivate PPAR gamma-responsive promoters. Previously, we showed that multidrug resistance proteins MRP1 and MRP3 attenuate cytotoxic and transactivating activities of 15-d-PGJ(2) in MCF7 breast cancer cells. Attenuation was glutathione-dependent and was associated with formation of the glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, and its active efflux by MRP. Here we have investigated whether the glutathione S-transferases (GST) can influence biological activities of 15-d-PGJ(2). MCF7 cells were stably transduced with human cytosolic GST isozymes M1a, A1, or P1a. These GSTs had no effect on 15-d-PGJ(2) cytotoxicity when expressed either alone or in combination with MRP1. However, expression of any of the three GSTs significantly inhibited 15-d-PGJ(2)-dependent transactivation of a PPAR gamma-responsive reporter gene. The degree of inhibition correlated with the level of GST expressed. Under physiologic conditions, the nonenzymatic rate of 15-d-PGJ(2) conjugation with glutathione was significant. Of the three GST isozymes, only GSTM1a-1a further stimulated the rate of 15-d-PGJ(2)-SG formation. Moreover, GSTM1a-1a rate enhancement was only a transient burst that was complete within 15 s. Hence, catalysis plays little, if any, role in GST inhibition of 15-d-PGJ(2)-dependent transactivation. In contrast, inhibition of transactivation was associated with strong GST/15-d-PGJ(2) interactions. Potent inhibition by 15-d-PGJ(2) and 15-d-PGJ(2)-SG of GST activity was observed with K(i) in the 0.15-2.0 microM range for the three GST isozymes, results suggesting avid associations between GST and 15-d-PGJ(2) or 15-d-PGJ(2)-SG. Electrospray ionization mass spectrometry (ESI/MS) studies revealed no stable adducts of GST and 15-d-PGJ(2) indicating that GST/15-d-PGJ(2) interactions are primarily noncovalent. These results are consistent with a mechanism of GST-mediated inhibition of transactivation in which GST binds 15-d-PGJ(2) and 15-d-PGJ(2)-SG thereby sequestering the ligands in the cytosol away from their nuclear target, PPAR gamma.     

CFTR, MDR1, and MRP1 immunolocalization in normal human nasal respiratory mucosa.

CFTR (cystic fibrosis transmembrane conductance regulator), MDR1 (multidrug resistance), and MRP1 (multidrug resistance-associated protein), members of the ABC transporter superfamily, possess multiple functions, particularly Cl(-), anion, and glutathione conjugate transport and cell detoxification. They are also hypothesized to have a number of complementary functions. It is generally accepted that data obtained from nasal mucosa can be extrapolated to lower airway cell physiology. The aim of the present study was to investigate by immunohistochemistry the differential localization of CFTR, MDR1, and MRP1 in the normal mucosa of 10 human nasal turbinates. In ciliated epithelial cells, CFTR was inconstantly expressed at the apical cell surface, intense membranous labeling was observed for MDR1, and intense cytoplasmic labeling was observed for MRP1. In the glands, a higher level of expression was observed on serous cells, at the apical surface (for CFTR), on lateral membranes (for MDR1), and with an intracytoplasmic distribution (for MRP1). In conclusion, CFTR, MDR1 and MRP1 are expressed in the epithelium and glands of the nasal respiratory mucosa, but with different patterns of expression. These results suggest major roles for CFTR, MDR1, and MRP1 in serous glandular cells and a protective function for MDR1 and MRP1 in respiratory ciliated cells. (J Histochem Cytochem 48:1215-1222, 2000)     

Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance

The membrane proteins mediating the ATP-dependent transport of lipophilic substances conjugated to glutathione, glucuronate, or sulfate have been identified as members of the multidrug resistance protein (MRP) family. Several isoforms of these conjugate export pumps with different kinetic properties and domain-specific localization in polarized human cells have been cloned and characterized. Orthologs of the human MRP isoforms have been detected in many different organisms. Studies in mutant rats lacking the apical isoform MRP2 (symbol ABCC2) indicate that anionic conjugates of endogenous and exogenous substances cannot exit from cells at a sufficient rate unless an export pump of the MRP family is present in the plasma membrane. Several mutations in the human MRP2 gene have been identified which lead to the absence of the MRP2 protein from the hepatocyte canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. Overexpression of recombinant MRP2 confers resistance to multiple chemotherapeutic agents. Because of its function in the terminal excretion of cytotoxic and carcinogenic substances, MRP2 as well as other members of the MRP family, play an important role in detoxification and chemoprevention.     

Glutathione and glutathione conjugate efflux from cultured liver cells

Efflux of glutathione (GSH) and GSH-conjugates from cultured rat liver epithelial cell lines; the non-tumorigenic ARL-15C₁ and the -glutamyl transpeptidase containing, tumorigenic ARL-16T₂, has been assessed under basal condition and during chronic treatment with 75 and 150 M ethacrynic acid (EA). The intracellular level of GSH increased in proportion to EA concentration during chronic exposure. The rates of GSH and GSH-EA conjugate efflux increased with intracellular GSH in both ARL cell lines.Glutathione-S-transferase activity measured with EA as substrate increased over the experimental time course after treatment with 150, but not 75 M EA. When intracellular GSH content was increased by treatment with the cysteine pro-drug, 2-L-oxothiazolidine 4-carboxylic acid, the rate of GSH efflux was increased, but not the rate of GS-EA conjugate export. Inhibition of -glutamyl transpeptidase by acivicin (AT-125) increased the GSH and GS-EA conjugate efflux rate in ARL-16T₂ cells by factors of approximately 2 and 15, respectively. Acivicin treatment of ARL-16T₂ cells chronically treated with EA elevated GSH efflux rate by 10-fold and GS-EA efflux by 40-fold versus control samples. These studies show that GSH and GSH conjugate efflux are accomplished as independently regulated processes. Efflux of GSH is enhanced by increased in racellular GSH, but increase in the conjugate transport rate requires the presence of the GSH conjugate. The response of the efflux process to treatment with a chronic GSH depleting agent was identical in two cell lines in which the metabolic fate of glutathione is known to differ fundamentally.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - GS-EA the glutathione conjugate of ethacrynic acid - EA ethacrynic acid - CDNB 1-chloro 2,4-dinitrobenzene - HBS HEPES buffered saline - OTC 2-L-oxothiazolidine 4-carboxylic acid - CYSSG cysteinyl-glutathione mixed disulfide - FDNB 1-fluoro-2,4-dinitrobenzene - GCS -glutamyl cysteine synthetase - GST glutathione-S-transferase - BCA bicinchoninic acid - SDS sodium dodecyl sulfate - PCA perchloric acid     

Polymorphisms of glutathione S-transferases M1, T1, P1 and A1 genes in the Tunisian population: an intra and interethnic comparative approach

Genetic polymorphisms in glutathione S-transferases (GSTs) genes might influence the detoxification activities of the enzymes predisposing individuals to cancer risk. Owing to the presence of these genetic variants, inter-individual and ethnic differences in GSTs detoxification capacity have been observed in various populations. Therefore, the present study was performed to determine the prevalence GSTM1 0/0, GSTT1 0/0, GSTP1 Ile(105)Val, and GSTA1 A/B polymorphisms in 154 healthy individuals from South Tunisia, and to compare them with those observed in North and Centre Tunisian populations and other ethnic groups. GSTM1 and GSTT1 polymorphisms were analyzed by a Multiplex-PCR approach, whereas GSTP1 and GSTA1 polymorphisms were examined by PCR-RFLP. The frequencies of GSTM10/0 and GSTT1 0/0 genotypes were 53.9% and 27.9%, respectively. The genotype distribution of GSTP1 was 47.4% (Ile/Ile), 40.9% (Ile/Val), and 11.7% (Val/Val). For GSTA1, the genotype distribution was 24.7% (A/A), 53.9% (A/B), and 21.4% (B/B). The combined genotypes distribution of GSTM1, GSTT1, GSTP1 and GSTA1 polymorphisms showed that thirty one of the 36 possible genotypes were present in our population; eight of them have a frequency greater than 5%. To the best of our knowledge, this is the first report of GSTs polymorphisms in South Tunisian population. Our findings demonstrate the impact of ethnicity and reveal a characteristic pattern for Tunisian population. The molecular studies in these enzymes provide basis for further epidemiological investigations in the population where these functional polymorphisms alter therapeutic response and act as susceptibility markers for various clinical conditions.