Transport Of Glutathione Conjugates Research Articles

The subgroup of 2′-hydroxy-flavonoids: Molecular diversity, mechanism of action, and anticancer properties

Flavonoids are abundant in nature, structurally very diversified and largely investigated. However, the subgroup of 2′-hydroxyflavonoids is much less known and not frequently studied. The present review identifies the major naturally-occurring and synthetic 2′-hydroxyflavonoid derivatives and discusses their structural characteristics and biological properties, with a focus on anticancer activities. The pharmacological properties of 2′-hydroxyflavone (2′-HF) and 2′-hydroxyflavanone (2′-HFa) are detailed. Upon binding to the Ral-interacting protein Rlip implicated in the transport of glutathione conjugates, 2′-HFa inhibits tumor cell proliferation and restrict tumor growth, in particular in breast cancer models. Among the synthetic derivatives, the characteristics of the anticancer product 2D08 (2′,3′,4′-trihydroxy flavone) are detailed to shed light on the molecular mechanism of action of this compound, as a regulator of protein SUMOylation. Inhibition of protein SUMOylation by 2D08 blocks cancer cell migration and invasion, and the compound greatly enhances the anticancer effects of conventional cytotoxic drugs like etoposide. The structural role of the 2′-hydroxyl group on the phenyl C-ring of the flavonoid is discussed, notably the capacity to engage intramolecular H-bonding interactions with the O1 atom on the B-ring of the chromone unit (or the oxygen of a 3-OH group when it is presents). The 2′-hydroxyl group of flavonoid appears as a regulator of the conformational freedom between the bicyclic A-B unit and the appended phenyl C-ring, favoring the planarity of the molecule. It is an essential group accounting for the biological properties of 2′-HF, 2′-HFa and structurally related compounds. This review shed light on 2′-hydroxyflavonoids to encourage their use and chemical development.

ABC Transporter-Mediated Transport of Glutathione Conjugates Enhances Seed Yield and Quality in Chickpea.

The identification of functionally relevant molecular tags is vital for genomics-assisted crop improvement and enhancement of seed yield, quality, and productivity in chickpea (Cicer arietinum). The simultaneous improvement of yield/productivity as well as quality traits often requires pyramiding of multiple genes, which remains a major hurdle given various associated epistatic and pleotropic effects. Unfortunately, no single gene that can improve yield/productivity along with quality and other desirable agromorphological traits is known, hampering the genetic enhancement of chickpea. Using a combinatorial genomics-assisted breeding and functional genomics strategy, this study identified natural alleles and haplotypes of an ABCC3-type transporter gene that regulates seed weight, an important domestication trait, by transcriptional regulation and modulation of the transport of glutathione conjugates in seeds of desi and kabuli chickpea. The superior allele/haplotype of this gene introgressed in desi and kabuli near-isogenic lines enhances the seed weight, yield, productivity, and multiple desirable plant architecture and seed-quality traits without compromising agronomic performance. These salient findings can expedite crop improvement endeavors and the development of nutritionally enriched high-yielding cultivars in chickpea.

Genome-wide identification and expression characterization of ABCC-MRP transporters in hexaploid wheat.

The ABCC multidrug resistance associated proteins (ABCC-MRP), a subclass of ABC transporters are involved in multiple physiological processes that include cellular homeostasis, metal detoxification, and transport of glutathione-conjugates. Although they are well-studied in humans, yeast, and Arabidopsis, limited efforts have been made to address their possible role in crop like wheat. In the present work, 18 wheat ABCC-MRP proteins were identified that showed the uniform distribution with sub-families from rice and Arabidopsis. Organ-specific quantitative expression analysis of wheat ABCC genes indicated significantly higher accumulation in roots (TaABCC2, TaABCC3, and TaABCC11 and TaABCC12), stem (TaABCC1), leaves (TaABCC16 and TaABCC17), flag leaf (TaABCC14 and TaABCC15), and seeds (TaABCC6, TaABCC8, TaABCC12, TaABCC13, and TaABCC17) implicating their role in the respective tissues. Differential transcript expression patterns were observed for TaABCC genes during grain maturation speculating their role during seed development. Hormone treatment experiments indicated that some of the ABCC genes could be transcriptionally regulated during seed development. In the presence of Cd or hydrogen peroxide, distinct molecular expression of wheat ABCC genes was observed in the wheat seedlings, suggesting their possible role during heavy metal generated oxidative stress. Functional characterization of the wheat transporter, TaABCC13 a homolog of maize LPA1 confirms its role in glutathione-mediated detoxification pathway and is able to utilize adenine biosynthetic intermediates as a substrate. This is the first comprehensive inventory of wheat ABCC-MRP gene subfamily.

Multidrug resistance-associated proteins are involved in the transport of the glutathione conjugates of the ultimate carcinogen of benzo[a]pyrene in human Caco-2 cells

A wide variety of contaminants are ingested through food, among them the pro-carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) that is resorbed and partially metabolized in the enterocytes of the small intestine. Previous in vitro studies have revealed that BP phenols are excreted as Phase II metabolites including glucuronides and sulfates. This export is mediated by the breast cancer resistance protein (ABCG2). The ultimate carcinogenic Phase I BP metabolite anti-BP-7,8-dihydrodiol-9,10-epoxide (BPDE) can be detoxified by glutathione conjugate formation catalyzed by glutathione S-transferases. In the present study, differentiated human intestinal Caco-2 cells were used as a model for the human small intestine to investigate the detoxification of BPDE and excretion of stereoisomeric glutathione conjugates in the presence of an inhibitor of the glutathione-cleaving enzyme γ-glutamyl transpeptidase at the cell surface. The results indicate that the glutathione conjugates of BPDE are formed and excreted mainly to the apical and to a minor extent to the basolateral side of polarized Caco-2 monolayers. Inhibition studies revealed that the multidrug resistance-associated proteins (ABCCs) are involved in the transport of BPDE glutathione conjugates. Stable ABCC1, ABCC2 and ABCC3 knockdown cell lines were generated, thus making it possible to demonstrate that ABCC1 mediates the basolateral and ABCC2 the apical excretion of BPDE glutathione conjugates. In conclusion, the ultimate carcinogen BPDE is detoxified via glutathione conjugation and subsequently excreted by Caco-2 cells in both apical and basolateral directions. This finding is equivalent to a transport into feces as well as blood system in the in vivo situation.

Luminal transport of thiol S-conjugates of methylmercury in isolated perfused rabbit renal proximal tubules

Lumen-to-cell transport, cellular accumulation, and toxicity of l-cysteine (Cys), glutathione (GSH) and N-acetylcysteine (NAC) S-conjugates of methylmercury (CH3Hg+) were evaluated in isolated, perfused rabbit proximal tubular segments. When these conjugates were perfused individually through the lumen of S2 segments of the proximal tubule it was found that Cys-S-CH3Hg and GSH-S-CH3Hg were transported avidly, while NAC-S-CH3Hg was transported minimally. In addition, 95% of the 203Hg taken up by the tubular cells was associated with precipitable proteins of the tubule, while very little was found in the acid-soluble cytosol. No visual cellular pathological changes were observed during 30min of study. Luminal uptake of Cys-S-CH3Hg was temperature-dependent and inhibited significantly by the amino acids l-methionine and l-cystine. Rates of luminal uptake of GSH-S-CH3Hg were twice as great as that of Cys-S-CH3Hg and uptake was inhibited significantly (74%) by the presence of acivicin. When 2,3-bis(sulfanyl)propane-1-sulfonate (DMPS) was added to the bathing or luminal fluid, luminal uptake of Cys-S-CH3Hg was diminished significantly. Overall, our data indicate that Cys-S-CH3Hg is likely a transportable substrate of one or more amino acid transporters (such as system B0,+ and system b0,+) involved in luminal absorption of l-methionine and l-cystine along the renal proximal tubule. In addition, GSH-S-CH3Hg appears to be degraded enzymatically to Cys-S-CH3Hg, which can then be taken up at the luminal membrane. By contrast NAC-S-CH3Hg and Cys-S-CH3Hg (in the presence of DMPS) are not taken up avidly at the luminal membrane of proximal tubular cells, thus promoting the excretion of CH3Hg+ into the urine.

Arsenic–glutathione conjugate transport by the human multidrug resistance proteins (MRPs/ABCCs)

Millions of people world-wide are chronically exposed to inorganic forms of the environmental toxicant arsenic in drinking water. This has led to a public health crisis because arsenic is a human carcinogen, and causes a myriad of other adverse health effects. In order to prevent and treat arsenic-induced toxicity it is critical to understand the cellular handling of this metalloid. A large body of literature describes the importance of the cellular tripeptide glutathione (γ-Glu-Cys-Gly,GSH/GS) in the excretion of arsenic. The triglutathione conjugate of arsenite [AsIII(GS)3] and the diglutathione conjugate of monomethylarsonous acid [MMAIII(GS)2] have been isolated from rat bile and mouse urine, and account for the majority of excreted arsenic, suggesting these are important transportable forms. The ATP-binding cassette (ABC) transporter proteins, multidrug resistance protein 1 (MRP1/ABCC1) and the related protein MRP2 (ABCC2), are thought to play an important role in arsenic detoxification through the cellular efflux of arsenic–GSH conjugates. Current knowledge on the cellular handling of arsenic with a special emphasis on the transport pathways of the arsenic–GSH conjugates AsIII(GS)3, MMAIII(GS)2, and dimethylarsenic glutathione DMAIII(GS), as well as, the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]− are reviewed.

RLIP76, a Glutathione-Conjugate Transporter, Plays a Major Role in the Pathogenesis of Metabolic Syndrome

PurposeCharacteristic hypoglycemia, hypotriglyceridemia, hypocholesterolemia, lower body mass, and fat as well as pronounced insulin-sensitivity of RLIP76−/− mice suggested to us the possibility that elevation of RLIP76 in response to stress could itself elicit metabolic syndrome (MSy). Indeed, if it were required for MSy, drugs used to treat MSy should have no effect on RLIP76−/− mice.Research Design and MethodsBlood glucose (BG) and lipid measurements were performed in RLIP76+/+ and RLIP76−/− mice, using Ascensia Elite Glucometer® for glucose and ID Labs kits for cholesterol and triglycerides assays. The ultimate effectors of gluconeogenesis are the three enzymes: PEPCK, F-1,6-BPase, and G6Pase, and their expression is regulated by PPARγ and AMPK. The activity of these enzymes was tested by protocols standardized by us. Expressions of RLIP76, PPARα, PPARγ, HMGCR, pJNK, pAkt, and AMPK were performed by Western-blot and tissue staining.ResultsThe concomitant activation of AMPK and PPARγ by inhibiting transport activity of RLIP76, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes like PEPCK, G6Pase and F-1,6-BP in RLIP76−/− mice, is a salient finding of our studies. The decrease in RLIP76 protein expression by rosiglitazone and metformin is associated with an up-regulation of PPARγ and AMPK.Conclusions/SignificanceAll four drugs, rosiglitazone, metformin, gemfibrozil and atorvastatin failed to affect glucose and lipid metabolism in RLIP76−/− mice. Studies confirmed a model in which RLIP76 plays a central role in the pathogenesis of MSy and RLIP76 loss causes profound and global alterations of MSy signaling functions. RLIP76 is a novel target for single-molecule therapeutics for metabolic syndrome.

The Rho1 GTPase Acts Together With a Vacuolar Glutathione S-Conjugate Transporter to Protect Yeast Cells From Oxidative Stress

Maintenance of redox homeostasis is critical for the survival of all aerobic organisms. In the budding yeast Saccharomyces cerevisiae, as in other eukaryotes, reactive oxygen species (ROS) are generated during metabolism and upon exposure to environmental stresses. The abnormal production of ROS triggers defense mechanisms to avoid the deleterious consequence of ROS accumulation. Here, we show that the Rho1 GTPase is necessary to confer resistance to oxidants in budding yeast. Temperature-sensitive rho1 mutants (rho1(ts)) are hypersensitive to oxidants and exhibit high accumulation of ROS even at a semipermissive temperature. Rho1 associates with Ycf1, a vacuolar glutathione S-conjugate transporter, which is important for heavy metal detoxification in yeast. Rho1 and Ycf1 exhibit a two-hybrid interaction with each other and form a bimolecular fluorescent complex on the vacuolar membrane. A fluorescent-based complementation assay suggests that the GTP-bound Rho1 associates with Ycf1 and that their interaction is enhanced upon exposure to hydrogen peroxide. The rho1(ts) mutants also exhibit hypersensitivity to cadmium, while cells carrying a deletion of YCF1 or mutations in a component of the Pkc1-MAP kinase pathway exhibit little or minor sensitivity to oxidants. We thus propose that Rho1 protects yeast cells from oxidative stress by regulating multiple downstream targets including Ycf1. Since both Rho1 and Ycf1 belong to highly conserved families of proteins, similar mechanisms may exist in other eukaryotes.

Targeting p53-Null Neuroblastomas through RLIP76

The search for p53-independent mechanism of cancer cell killing is highly relevant to pediatric neuroblastomas, where successful therapy is limited by its transformation into p53-mutant and a highly drug-resistant neoplasm. Our studies on the drug-resistant p53-mutant as compared with drug-resistant p53 wild-type neuroblastoma revealed a novel mechanism for resistance to apoptosis: a direct role of p53 in regulating the cellular concentration of proapoptotic alkenals by functioning as a specific and saturable allosteric inhibitor of the alkenal-glutathione conjugate transporter, RLIP76. The RLIP76-p53 complex was showed by both immunoprecipitation analyses of purified proteins and immunofluorescence analysis. Drug transport studies revealed that p53 inhibited both basal and PKCα-stimulated transport of glutathione conjugates of 4HNE (GSHNE) and doxorubicin. Drug resistance was significantly greater for p53-mutant as compared with p53 wild-type neuroblastoma cell lines, but both were susceptible to depletion of RLIP76 by antisense alone. In addition, inhibition of RLIP76 significantly enhanced the cytotoxicity of cisplatin. Taken together, these studies provide powerful evidence for a novel mechanism for drug and apoptosis resistance in p53-mutant neuroblastoma, based on a model of regulation of p53-induced apoptosis by RLIP76, where p53 is a saturable and specific allosteric inhibitor of RLIP76, and p53 loss results in overexpression of RLIP76; thus, in the absence of p53, the drug and glutathione-conjugate transport activities of RLIP76 are enhanced. Most importantly, our findings strongly indicate RLIP76 as a novel target for therapy of drug-resistant and p53-mutant neuroblastoma.

Abstract 5547: Glutathione-conjugate transport by RLIP76 is required for clathrin-dependent endocytosis and chemical carcinogenesis

Abstract Targeted depletion of the RALBP1 encoded 76 kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancer without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad-spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport-activity is required for clathrin-dependent endocytosis (CDE) that regulates all ligand-receptor signaling and that RLIP76 is required not only for survival of cancer cells, but also for their very existence. We studied RLIP76 mutant proteins and the functional consequences of their expression into RLIP76−/− MEFs which identified key residues for GS-E binding in RLIP76, established the requirement of RLIP76-mediated GS-E transport for CDE and demonstrated a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 near completely blocked signaling down-stream of EGF in a CDE-dependent manner and Wnt5a signaling in a CDE-independent manner. The seminal prediction of this hypothesis, that RLIP76−/− mice will be deficient in chemical neoplasia, was confirmed. Benzo[a]pyrene, dimethylbenzanthracene and phorbol esters are ineffective in causing neoplasia in RLIP76−/−. PMA-induced skin carcinogenesis in RLIP76+/+ mouse was suppressed completely by depletion of either PKCα or RLIP76 by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76−/− mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76−/− mice. In RLIP76−/− mice, p53, p38, and JNK activation did not occur in response to either carcinogen. Our findings demonstrate a fundamental role of RLIP76 in chemical carcinogenesis. (Supported in part by NIH grant CA 77495 (SA), CA 155350, Cancer Research Foundation of North Texas, and Institute for Cancer Research & the Joe & Jessie Crump Fund for Medical Education (SS)) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5547. doi:10.1158/1538-7445.AM2011-5547

Abstract 4334: Targeting p53 null neuroblastomas through RLIP76

Abstract The search for p53-independent mechanism of cancer cell killing is highly relevant to pediatric neuroblastomas, where successful therapy is limited by its transformation into p53 mutant and a highly drug-resistant neoplasm. Our studies on the drug-resistant p53 mutant as compared with drug-resistant p53 wild-type neuroblastoma revealed a novel mechanism for resistance to apoptosis: a direct role of p53 in regulating the cellular concentration of pro-apoptotic alkenals by functioning as a specific and saturable allosteric inhibitor of the alkenal-glutathione-conjugate transporter, RLIP76. The RLIP76-p53 complex was demonstrated both by using immuno-precipitation analyses of purified proteins as well as by immuno-fluorescence analysis. Drug transport studies revealed that p53 inhibited both basal and PKCα stimulated transport of glutathione-conjugates of 4HNE (GS-HNE) and cisplatin. Drug resistance was significantly greater for p53 mutant as compared with p53 wild-type neuroblastoma cell lines, but both were susceptible to depletion of RLIP76 by antisense alone. In addition, inhibition of RLIP76 significantly enhanced the cytotoxicity of cisplatin. Taken together, these studies provide powerful evidence for a novel mechanism for drug and apoptosis resistance in p53 mutant neuroblastoma, based on a model of regulation of p53 induced apoptosis by RLIP76, where p53 is a saturable and specific allosteric inhibitor of RLIP76, and p53 loss results in over-expression of RLIP76; thus, in the absence of p53, the drug and glutathione-conjugate transport activities of RLIP76 are enhanced. Most importantly, our findings strongly indicate RLIP76 as a novel target for therapy of drug-resistant and p53 mutant neuroblastoma. (Supported in part by NIH grant CA 77495 (SA), CA 155350, Cancer Research Foundation of North Texas, and Institute for Cancer Research & the Joe & Jessie Crump Fund for Medical Education (SSS)) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4334. doi:10.1158/1538-7445.AM2011-4334

Glutathione-conjugate transport by RLIP76 is required for clathrin-dependent endocytosis and chemical carcinogenesis.

Targeted depletion of the RALBP1-encoded 76-kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancers without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport activity is required for clathrin-dependent endocytosis (CDE), which regulates all ligand-receptor signaling, and that RLIP76 is required not only for survival of cancer cells but also for their very existence. We studied RLIP76 mutant proteins and the functional consequences of their expression into RLIP76(-/-) MEFs, identified key residues for GS-E binding in RLIP76, established the requirement of RLIP76-mediated GS-E transport for CDE, and showed a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 nearly completely blocked signaling downstream of EGF in a CDE-dependent manner and Wnt5a signaling in a CDE-independent manner. The seminal prediction of this hypothesis-RLIP76(-/-) mice will be deficient in chemical neoplasia-was confirmed. Benzo[a]pyrene, dimethylbenzanthracene, and phorbol esters are ineffective in causing neoplasia in RLIP76(-/-). PMA-induced skin carcinogenesis in RLIP76(+/+) mouse was suppressed completely by depletion of either PKCα or RLIP76 by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76(-/-) mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76(-/-) mice. In RLIP76(-/-) mice, p53, p38, and JNK activation did not occur in response to either carcinogen. Our findings show a fundamental role of RLIP76 in chemical carcinogenesis.

The role of multixenobiotic transporters in predatory marine molluscs as counter-defense mechanisms against dietary allelochemicals

Multixenobiotic transporters have been extensively studied for their ability to modulate the disposition and toxicity of pharmacological agents, yet their influence in regulating the levels of dietary toxins within marine consumers has only recently been explored. This study presents functional and molecular evidence for multixenobiotic transporter-mediated efflux activity and expression in the generalist gastropod Cyphoma gibbosum, and the specialist nudibranch Tritonia hamnerorum, obligate predators of chemically defended gorgonian corals. Immunochemical analysis revealed that proteins with homology to permeability glycoprotein (P-gp) were highly expressed in T. hamnerorum whole animal homogenates and localized to the apical tips of the gut epithelium, a location consistent with a role in protection against ingested prey toxins. In vivo dye assays with specific inhibitors of efflux transporters demonstrated the activity of P-gp and multidrug resistance-associated protein (MRP) families of ABC transporters in T. hamnerorum. In addition, we identified eight partial cDNA sequences encoding two ABCB and two ABCC proteins from each molluscan species. Digestive gland transcripts of C. gibbosum MRP-1, which have homology to vertebrate glutathione-conjugate transporters, were constitutively expressed regardless of gorgonian diet. This constitutive expression may reflect the ubiquitous presence of high affinity substrates for C. gibbosum glutathione transferases in gorgonian tissues likely necessitating export by MRPs. Our results suggest that differences in multixenobiotic transporter expression patterns and activity in molluscan predators may stem from the divergent foraging strategies of each consumer.

Rlip76 transports sunitinib and sorafenib and mediates drug resistance in kidney cancer

RLIP76 is a stress-responsive membrane protein implicated in the regulation of multiple cellular signaling pathways. It represents the predominant glutathione-conjugate (GS-E) transporter in cells. We have shown that RLIP76 plays a crucial role in defending cancer cells from radiation and chemotherapeutic toxin-mediated apoptosis, and that its inhibition by antibodies or depletion by siRNA or antisense causes apoptosis in a number of cancer cell types. We demonstrated for the first time that the striking anti-neoplastic effects with no evident toxicity in terms of either weight loss or metabolic effects are also demonstrable for the antibody, antisense and siRNA in a renal cell xenografts model of Caki-2 cells (Singhal et al., Cancer Res., 2009, 69: 4244). Present studies were performed to determine if RLIP76 targeting is more broadly applicable in other kidney cancer cell lines, to compare the signaling effects of RLIP76 antisense with kinase inhibitors used in treatment of renal cell carcinoma, and to determine whether kinase inhibitors were substrates for transport by RLIP76. Results of these studies show that sorafenib as well as sunitinib are substrates for transport by RLIP76 thus are competitive inhibitors of GS-E transport. Furthermore, kinase inhibition in the ERK as well as PI3K pathways by RLIP76 depletion is more profound and consistent and is more widely apparent in a number of renal carcinoma cell lines. These studies offer strong support for our overall hypothesis that RLIP76 is an overarching anti-apoptosis mechanism that, if inhibited, can be more broadly effective in the treatment of renal cell carcinoma.

Multidrug resistance‐associated proteins and implications in drug development

1. The multidrug resistance-associated proteins (MRPs) belong to the ATP-binding cassette superfamily (ABCC family) of transporters that are expressed differentially in the liver, kidney, intestine and blood-brain barrier. There are nine human MRPs that transport a structurally diverse array of endo- and xenobiotics as well as their conjugates. 2. Multidrug resistance-associated protein 1 can be distinguished from MRP2 and MRP3 by its higher affinity for leukotriene C(4). Unlike MRP1, MRP2 functions in the extrusion of endogenous organic anions, such as bilirubin glucuronide and certain anticancer agents. In addition to the transport of glutathione and glucuronate conjugates, MRP3 has the additional capability of mediating the transport of monoanionic bile acids. 3. Both MRP4 and MRP5 are able to mediate the transport of cyclic nucleotides and confer resistance to certain antiviral and anticancer nucleotide analogues. Hereditary deficiency of MRP6 results in pseudoxanthoma elasticum. In the body, MRP6 is involved in the transport of glutathione conjugates and the cyclic pentapeptide BQ123. 4. Various MRPs show considerable differences in tissue distribution, substrate specificity and proposed physiological function. These proteins play a role in drug disposition and excretion and thus are implicated in drug toxicity and drug interactions. Increased efflux of natural product anticancer drugs and other anticancer agents mediated by MRPs from cancer cells is associated with tumour resistance. 5. A better understanding of the function and regulating mechanisms of MRPs could help minimize and avoid drug toxicity and unfavourable drug-drug interactions, as well as help overcome drug resistance.

Physiological and Pharmacological Significance of Glutathione-Conjugate Transport

Transport of the glutathione conjugates (GS-E) of electrophilic compounds generated during biotransformation of drugs and environmental pollutants is central to the mechanisms of defense against oxidative/electrophilic stress. In recent years emphasis has been placed on ATP-binding cassette (ABC) transport proteins in the transport of GS-E and their involvement in the detoxification mechanisms, including drug resistance. Recent studies, however, suggested that the majority of GS-E transport in human and rodent cells is mediated by a non-ABC, multifunctional stress-response protein, RLIP76 or RalBP1 (ral-binding GTPase activating protein 1), which also functions as an effector in the Ral-Ras-Rho signaling pathway. In this review, after briefly describing the major discoveries in the field of glutathione (GSH)-conjugate transport, recent findings are presented on the role of RLIP76 in ATP-dependent transport of GS-E, and the relevance of this transport process to the mechanisms of toxicity of xenobiotics, radiation, and endogenous electrophilic toxicants is described. Furthermore, recent studies suggesting a link between RLIP76 mediated GS-E transport and cell cycle signaling are presented.

Influence of luminal monosaccharides on secretion of glutathione conjugates from rat small intestine in vitro

Intestinal efflux transporters can significantly reduce the absorption of the drug after peroral application. In this work we studied secretion of glutathione conjugates triggered by glucose at the luminal side of the intestine. Glucose stimulated secretion of DNPSG, NEMSG and CDNB. We used some different monosaccharides and determined that glucose, galactose and α-methylglucopyranoside trigger the secretion, while mannitol and fructose do not. We concluded that interaction with SGLT transporter is the key process necessary for this triggering. To determine which of possible glutathione conjugate transporters (MRP2, MRP4, BCRP or RLIP76) is responsible for the secretion of glutathione conjugates, we used benzbromarone, a MRP inhibitor, and sulfanitran and furosemide, two allosteric MRP2 activators. Benzbromarone inhibited glucose stimulated DNPSG secretion, while allosteric activators additionally increased the secretion. We concluded that MRP2 transporter is related to glucose stimulated DNPSG secretion. Regarding the work of Kubitz et al. we tested the effect of changed medium osmolarity on DNPSG transport and determined that hypoosmolar conditions trigger secretion of DNPSG. These findings suggest that intestinal MRP2 activity has no basal level, but can be stimulated by hypoosmolarity and SGLT transport.

Plant Vacuolar ATP-binding Cassette Transporters That Translocate Folates and Antifolates in Vitro and Contribute to Antifolate Tolerance in Vivo

The vacuoles of pea (Pisum sativum) leaves and red beet (Beta vulgaris) storage root are major sites for the intracellular compartmentation of folates. In the light of these findings and preliminary experiments indicating that some plant multidrug resistance-associated protein (MRP) subfamily ATP-binding cassette transporters are able to transport compounds of this type, the Arabidopsis thaliana vacuolar MRP, AtMRP1 (AtABCC1), and its functional equivalent(s) in vacuolar membrane vesicles purified from red beet storage root were studied. In so doing, it has been determined that heterologously expressed AtMRP1 and its equivalents in red beet vacuolar membranes are not only competent in the transport of glutathione conjugates but also folate monoglutamates and antifolates as exemplified by pteroyl-l-glutamic acid and methotrexate (MTX), respectively. In agreement with the results of these in vitro transport measurements, analyses of atmrp1 T-DNA insertion mutants of Arabidopsis ecotypes Wassilewskia and Columbia disclose an MTX-hypersensitive phenotype. atmrp1 knock-out mutants are more sensitive than wild-type plants to growth retardation by nanomolar concentrations of MTX, and this is associated with impaired vacuolar antifolate sequestration. The vacuoles of protoplasts isolated from the leaves of Wassilewskia atmrp1 mutants accumulate 50% less [(3)H]MTX than the vacuoles of protoplasts from wild-type plants when incubated in media containing nanomolar concentrations of this antifolate, and vacuolar membrane-enriched vesicles purified from the mutant catalyze MgATP-dependent [(3)H]MTX uptake at only 40% of the capacity of the equivalent membrane fraction from wild-type plants. AtMRP1 and its counterparts in other plant species therefore have the potential for participating in the vacuolar accumulation of folates and related compounds.

How Plants Cope with Foreign Compounds. Translocation of xenobiotic glutathione conjugates in roots of barley (Hordeum vulgare) (9 pp)

Numerous herbicides and xenobiotic organic pollutants are detoxified in plants to glutathione conjugates. Following this enzyme catalyzed reaction, xenobiotic GS-conjugates are thought to be compartmentalized in the vacuole of plant cells. In the present study, evidence is presented from experiments with roots of barley (Hordeum vulgare cv. Cherie) that part of these conjugates will undergo long range transport in plants, rather than be stored in the vacuole. To our knowledge, this is the first report about the unidirectional long-range transport of xenobiotic conjugates in plants and the exsudation of a glutathione conjugate from the root tips. This could mean that plants possess an excretion system for unwanted compounds giving them similar advantages as animals. Barley plants (Hordeum vulgare) were grown in Petri dishes soaked with tap water in the greenhouse. Fluorescence Microscopy. Roots of barley seedlings were cut under water, and the end at which the pesticides were applied was fixed in an aperture with a thin latex foil and transferred into a drop of water on a cover slide. The cover slide was fixed in a measuring chamber on the stage of an inverse fluorescence microscope (Zeiss Axiovert 100). Monobromo- and Monochlorobimane, two model xenobiotics that are conjugated rapidly in plant cells with glutathione, hereby forming fluorescent metabolites, were used as markers. Their transport in the root could be followed with high time resolution. Spectrometric enzyme assay. Glutathione S-transferase (GST) activity was determined in the protein extracts following established methods. Aliquots of the enzyme extract were incubated with 1-chloro-2,4-dinitrobenzene (CDNB), or monochlorobimane. Controls lacking enzyme or GSH were measured. Pitman chamber experiments. Ten days old barley plants or detached roots were inserted into special incubation chambers, either complete with tips or decapitated, as well as 10 days old barley plants without root tips. Compartment A was filled with a transport medium and GSH conjugate or L-cysteine conjugate. Compartments B and C contained sugar free media. Samples were taken from the root tip containing compartment C and the amount of conjugate transported was determined spectro-photometrically. Results. The transport in roots is unidirectional towards the root tips and leads to exsudation of the conjugates at rates between 20 and 200 nmol min(-1). The microscopic studies have been complemented by transport studies in small root chambers and spectroscopic quantification of dinitrobenzene-conjugates. The latter experiments confirm the microscopic studies. Furthermore it was shown that glutathione conjugates are transported at higher rates than cysteine conjugates, despite of their higher molecular weights. This observation points to the existence of glutathione specific carriers and a specific role of glutathione in the root. It can be assumed that long distance transport of glutathione conjugates within the plant proceeds like GSH or amino acid transport in both, phloem and xylem. The high velocity of this translocation of the GS-X is indicative of an active transport. For free glutathione, a rapid transport-system is essential because an accumulation of GSH in the root tip inhibits further uptake of sulfur. Taking into account that all described MRP transporters and also the GSH plasmalemma ATPases have side activities for glutathione derivatives and conjugates, co-transport of these xenobiotic metabolites seems credible. On the other hand, when GS-B was applied to the root tips from the outside, no significant uptake was observed. Thus it can be concluded that only those conjugates can be transported in the xylem which are formed inside the root apex. Having left the root once, there seems to be no return into the root vessels, probably because of a lack of inward directed transporters. Plants seem to possess the capability to store glutathione conjugates in the vacuole, but under certain conditions, these metabolites might also undergo long range transport, predominantly into the plant root. The transport seems dependent on specific carriers and is unidirectional, this means that xenobiotic conjugates from the rhizosphere are not taken up again. The exudation of xenobiotic metabolites offers an opportunity to avoid the accumulation of such compounds in the plant. The role of glutathione and glutathione related metabolites in the rhizosphere has not been studied in any detail, and only scattered data are available on interactions between the plant root and rhizosphere bacteria that encounter such conjugates. The final fate of these compounds in the root zone has also not been addressed so far. It will be interesting to study effects of the exsuded metabolites on the biology of rhizosphere bacteria and fungi.

Mitogenic Responses of Vascular Smooth Muscle Cells to Lipid Peroxidation-derived Aldehyde 4-Hydroxy-trans-2-nonenal (HNE): ROLE OF ALDOSE REDUCTASE-CATALYZED REDUCTION OF THE HNE-GLUTATHIONE CONJUGATES IN REGULATING CELL GROWTH

Products of lipid peroxidation such as 4-hydroxy-trans-2-nonenal (HNE) trigger multiple signaling cascades that variably affect cell growth, differentiation, and apoptosis. Because glutathiolation is a significant metabolic fate of these aldehydes, we tested the possibility that the bioactivity of HNE depends upon its conjugation with glutathione. Addition of HNE or the cell-permeable esters of glutathionyl-4-hydroxynonenal (GS-HNE) or glutathionyl-1,4-dihydroxynonene (GS-DHN) to cultures of rat aortic smooth muscle cells stimulated protein kinase C, NF-kappaB, and AP-1, and increased cell growth. The mitogenic effects of HNE, but not GS-HNE or GS-DHN, were abolished by glutathione depletion. Pharmacological inhibition or antisense ablation of aldose reductase (which catalyzes the reduction of GS-HNE to GS-DHN) prevented protein kinase C, NF-kappaB, and AP-1 stimulation and the increase in cell growth caused by HNE and GS-HNE, but not GS-DHN. The growth stimulating effect of GS-DHN was enhanced in cells treated with antibodies directed against the glutathione conjugate transporters RLIP76 (Ral-binding protein) or the multidrug resistance protein-2. Overexpression of RLIP76 abolished the mitogenic effects of HNE and its glutathione conjugates, whereas ablation of RLIP76 using RNA interference promoted the mitogenic effects. Collectively, our findings suggest that the mitogenic effects of HNE are mediated by its glutathione conjugate, which has to be reduced by aldose reductase to stimulate cell growth. These results raise the possibility that the glutathione conjugates of lipid peroxidation products are novel mediators of cell signaling and growth.