Interactions of mefloquine with ABC proteins, MRP1 (ABCC1) and MRP4 (ABCC4) that are present in human red cell membranes

Human erythrocyte membranes express the multidrug resistance-associated proteins, MRP1, MRP4 and 5, that collectively can efflux oxidised glutathione, glutathione conjugates and cyclic nucleotides. It is already known that the quinoline derivative, MK-571, is a potent inhibitor of MRP-mediated transport. We here examine whether the quinoline-based antimalarial drugs, amodiaquine, chloroquine, mefloquine, primaquine, quinidine and quinine, also interact with erythrocyte MRPs with consequences for their access to the intracellular parasites or for efflux of oxidised glutathione from infected cells. Using inside-out vesicles prepared from human erythrocytes we have shown that mefloquine and MK-571 inhibit transport of 3 microM [(3)H]DNP-SG known to be mediated by MRP1 (IC(50) 127 and 1.1 microM, respectively) and of 3.3 microM [(3)H]cGMP thought but not proven to be mediated primarily by MRP4 (IC(50) 21 and 0.41 microM). They also inhibited transport in membrane vesicles prepared from tumour cells expressing MRP1 or MRP4 and blocked calcein efflux from MRP1-overexpressing cells and BCECF efflux from MRP4-overexpressing cells. Both stimulated ATPase activity in membranes prepared from MRP1 and MRP4-overexpressing cells and inhibited activity stimulated by quercetin or PGE(1), respectively. Neither inhibited [alpha-(32)P]8-azidoATP binding confirming that the interactions are not at the ATP binding site. These results demonstrate that mefloquine and MK-571 both inhibit transport of other substrates and stimulate ATPase activity and thus may themselves be substrates for transport. But at concentrations achieved clinically mefloquine is unlikely to affect the MRP1-mediated transport of GSSG across the erythrocyte membrane.     

Transport of glutathione and glutathione conjugates by MRP1

Glutathione (GSH)-conjugated xenobiotics and GSH-conjugated metabolites (e.g. the cysteinyl leukotriene C4) must be exported from the cells in which they are formed before they can be eliminated from the body or act on their cellular targets. This efflux is often mediated by the multidrug resistance protein 1 (MRP1) transporter, which also confers drug resistance to tumour cells and can protect normal cells from toxic insults. In addition to drugs and GSH conjugates, MRP1 exports GSH and GSH disulfide, and might thus have a role in cellular responses to oxidative stress. The transport of several drugs and conjugated organic anions by MRP1 requires the presence of GSH, but it is not well understood how GSH (and its analogues) enhances transport. Site-directed mutagenesis studies and biophysical analyses have provided important insights into the structural determinants of MRP1 that influence GSH and GSH conjugate binding and transport.     

Intracellular glutathione regulates taurocholate transport in HepG2 cells

The hepatic organic anion transporter 1, Oatp1, was recently demonstrated to function as a GSH exchanger, indicating that hepatic uptake of drugs and xenobiotics may be sensitive to intracellular GSH levels. The present study characterized taurocholate uptake and efflux mechanisms in HepG2 cells and the effects of intracellular GSH on these transport processes. Taurocholate uptake into HepG2 cells was Na(+)-independent, saturable ( K(m) = 82 +/- 16 microM), and was cis-inhibited by bromosulfophthalein and some bile acids. Intracellular GSH depletion inhibited 3H-taurocholate uptake, and, conversely, the release of GSH from HepG2 cells was stimulated in the presence of extracellular taurocholate and other bile acids, consistent with a role for intracellular GSH in stimulating organic anion uptake. Interestingly, efflux of 3H-taurocholate from HepG2 cells was also sensitive to intracellular GSH concentration: efflux was inhibited in cells with lower intracellular GSH and stimulated in cells with higher GSH. RT-PCR analysis revealed that OATP-A, OATP-D, OATP-E, OATP-8, MRP1, MRP2, and MRP3 are expressed in HepG2 cells but that their expression is not altered by the maneuvers used to lower or raise intracellular GSH. These results provide direct evidence that intracellular GSH levels modulate both uptake and efflux of taurocholate and suggest that GSH plays a regulatory role in the hepatobiliary transport of potentially toxic organic compounds.     

Kinetics of glutathione and daunorubicin efflux from multidrug resistance protein overexpressing small-cell lung cancer cells.

The present study examined how the multidrug resistance protein (MRP1), which is an ATP-dependent anionic conjugate transporter, also mediates the transport of reduced glutathione (GSH) and the co-transport of the cationic drug, daunorubicin, with GSH in living GLC4/Adr cells. To obtain information on the affinity of GSH for the multidrug resistance protein in GLC4/Adr cells, we investigated the GSH concentration dependence of the ATP-dependent GSH efflux. The intracellular GSH concentration was modulated by preincubation of the cells with 25 microM buthionine sulfoximine, an inhibitor of GSH synthetase, for 0-24 h. The transport of GSH was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. Fitting of the obtained data according to the Michaelis-Menten equation revealed a Km of 3.4+/-1.4 mM and a Vmax of 1.5+/-0.2x10(-18) mol/cell/s. The ATP-dependent transport of GSH was inhibited by 3-([[3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl]-[(3-dimethylamino-3-oxopropyl)-thio]-methyl]thio)propanoic acid (MK571), with 50% inhibition being obtained with 1.4 microM MK571. We investigated the GSH concentration dependence of the MRP1-mediated ATP-dependent transport of daunorubicin under conditions where the transport of daunorubicin became saturated. The daunorubicin transport was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. We were therefore in the situation where GSH acted as an activator: its presence was necessary for the binding and transport of daunorubicin by MRP1. However, GSH was also transported by the multidrug resistance protein. The concentration of GSH that gave half the maximal rate of daunorubicin efflux was 2.1+/-0.8 mM, very similar to the Km value obtained for GSH. In conclusion, the rate of daunorubicin efflux, under conditions where the transport of daunorubicin became saturated, and the rate of GSH efflux determined at any intracellular concentration of GSH were very similar, yielding a 1:1 stoichiometry with respect to GSH and daunorubicin transport. These results support a model in which daunorubicin is co-transported with GSH.     

cGMP (guanosine 3',5'-cyclic monophosphate) transport across human erythrocyte membranes

Human erythrocytes produce cGMP that can be eliminated by phosphodiesterases or active efflux transporters. The efflux can be studied under controlled conditions as ATP-dependent uptake into inside-out membrane vesicles. However, widely differing values for the transport rates have been reported. We have here examined factors that influence the uptake rates measured and thus may explain these discrepancies. Both the ionic composition of the buffer used during uptake and the mode of vesicle preparation were found to affect the observed transport rates. Furthermore it was apparent that different blood donors expressed on their erythrocytes different amounts of both MRP4 and MRP5, transporters that have been putatively linked to cGMP efflux across erythrocyte membranes. These differences in expression were reflected in differences in rates of cGMP uptake into inside-out erythrocyte membrane vesicles. Calculations based on the transport rates observed using vesicles suggest that efflux may be the principal means for eliminating cGMP from human erythrocytes.     

Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins

The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport GSH from the cytosol into the vacuole, whereas Hgt1p mediates GSH uptake across the plasma membrane. Because transport is a key step in GSH homeostasis and is intimately linked to its biological functions, GSH export proteins are likely to modulate essential cellular functions.     

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.     

Transport of prostaglandin E1 across rat erythrocyte membrane

In this study erythrocyte transport of prostaglandin E1 (PGE1) was investigated by employing inside-out membrane vesicles prepared from rat erythrocytes. The uptake of [3H]PGE1 in the presence of ATP was significantly higher than that of AMP, suggesting the involvement of an ATP-dependent efflux system in PGE1 transport across the erythrocyte membrane. Coincubation of glutathione with ATP further stimulated the uptake of [3H]PGE1. The uptake of [3H]PGE1 in the presence of ATP and glutathione was temperature-sensitive, and various eicosanoids including PGE2 and PGF2alpha decreased the uptake. Multidrug resistance-associated protein (MRP) 4 substrates/inhibitors including methotrexate, indomethacin, taurocholic acid and indocyanine green significantly inhibited [3H]PGE1 uptake. Western blot analysis revealed that Mrp4 is expressed in rat erythrocyte membrane. These results suggest that the release of PGE1 from the erythrocyte into the blood circulation may be mediated by ATP-dependent efflux pump(s) such as Mrp4.     

Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy

The nine multidrug resistance proteins (MRPs) represent the major part of the 12 members of the MRP/CFTR subfamily belonging to the 48 human ATP-binding cassette (ABC) transporters. Cloning, functional characterization, and cellular localization of most MRP subfamily members have identified them as ATP-dependent efflux pumps with a broad substrate specificity for the transport of endogenous and xenobiotic anionic substances localized in cellular plasma membranes. Prototypic substrates include glutathione conjugates such as leukotriene C(4) for MRP1, MRP2, and MRP4, bilirubin glucuronosides for MRP2 and MRP3, and cyclic AMP and cyclic GMP for MRP4, MRP5, and MRP8. Reduced glutathione (GSH), present in living cells at millimolar concentrations, modifies the substrate specificities of several MRPs, as exemplified by the cotransport of vincristine with GSH by MRP1, or by the cotransport of GSH with bile acids or of GSH with leukotriene B(4) by MRP4.The role of MRP subfamily members in pathophysiology may be illustrated by the MRP-mediated release of proinflammatory and immunomodulatory mediators such as leukotrienes and prostanoids. Pathophysiological consequences of many genetic variants leading to a lack of functional MRP protein in the plasma membrane are observed in the hereditary MRP2 deficiency associated with conjugated hyperbilirubinemia in Dubin-Johnson syndrome, in pseudoxanthoma elasticum due to mutations in the MRP6 (ABCC6) gene, or in the type of human earwax and osmidrosis determined by single nucleotide polymorphisms in the MRP8 (ABCC8) gene. The hepatobiliary and renal elimination of many drugs and their metabolites is mediated by MRP2 in the hepatocyte canalicular membrane and by MRP4 as well as MRP2 in the luminal membrane of kidney proximal tubules. Therefore, inhibition of these efflux pumps affects pharmacokinetics, unless compensated by other ATP-dependent efflux pumps with overlapping substrate specificities.     

Kinetic analysis of fluorescein and dihydrofluorescein effluxes in tumour cells expressing the multidrug resistance protein,MRP1

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of two transporters the P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP1) which actively pump out multiple chemically unrelated substrates across the plasma membrane. A clear distinction in the mechanism of translocation of substrates by MRP1 or P-gp is indicated by the finding that, in most of cases, the MRP1-mediated transport of substrates is inhibited by depletion of intracellular glutathione (GSH), which has no effect on their P-gp-mediated transport. The aim of the present study was to quantitatively characterise the transport of anionic compounds dihydrofluorescein and fluorescein (FLU). We took advantage of the intrinsic fluorescence of FLU and performed a flow cytometric analysis of dye accumulation in the wild-type drug sensitive GLC4 that do not express MRP1 and its MDR subline which display high level of MRP1. The measurements were made in real time using intact cells. The kinetics parameters, k(a)=V(M)/K(m), which is a measure of the efficiency of the transporter-mediated efflux of a substrate, was very similar for the two FLU analogues. They were highly comparable with values for k(a) of other negatively charged substrates, such as GSH and calcein. The active efflux of both FLU derivatives was inhibited by GSH depletion.     

Multidrug Resistance Associated Protein 1 together with Glutathione Plays a Protective Role against 4-Hydroxy-2-nonenal-Induced Oxidative Stress in Bovine Aortic Endothelial Cells

4-Hydroxy-2-nonenal (HNE), an aldehyde produced by lipid peroxidation, induces cytotoxicity and oxidative stress. Glutathione (GSH) protects against the cytotoxicity of HNE. However, the protective mechanism of GSH has not been fully examined. We examined the protective role played by the relationship between GSH and multidrug resistance associated protein 1 (MRP1) against the HNE-induced oxidative stress in bovine aortic endothelial cells (BAECs). HNE induced the loss of viability of BAECs. Exogenous GSH, which is membrane-impermeable, prevented the loss of viability induced by HNE by inhibiting HNE uptake in BAECs, probably due to the formation of the HNE-SG complex in the extracellular space. We demonstrated that HNE induced the expression of MRP1 protein, which can transport the HNE-SG complex. The induction of MRP1 protein expression by HNE disappeared in BAECs pretreated with L-buthionine sulfoximine, a GSH-depleting agent. This result suggests that HNE, together with intracellular GSH, contributes to the regulation of MRP1 protein expression. Moreover, we found that MK571, an MRP1 inhibitor, promoted the HNE-induced oxidative stress and cell death. Taken together, these findings suggest that MRP1, together with GSH, plays a protective role against the HNE-induced oxidative stress in BAECs.     

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.     

Non-destructive micromethod for MRP1 functional assay in human lung tumor cells

Defense against toxic endo- and xenobiotics is a major concern of all living species and ABC transporters play a vital role in this defense system. Multidrug resistance associated proteins 1 (MRP1) is a cellular detoxifying factor supposed to transport a wide range of compounds across cell membranes either as GSH conjugates or as co-transport accompanying glutathione transposition. The cellular localization of MRP1 is a determining factor whether the transport function can take place. In this study we have undertaken experiments on the transport activity of MRP1 in cultured human lung tumor cells in order to check whether MRP1 is expressed as a functionally active protein. For this purpose we have adapted a quantitative fluorescence imaging assay to conditions where a small number of attached cells should be repeatedly measured by a non-destructive method. In cultured A549, H358 and H322 cells MRP1 is located in the cell membrane as observed by immunocytochemistry. Efflux of 5,6-carboxy-2′-7′-dichloro-fluorescein (CDF) from lung cells was sensitive toward the MRP1 inhibitor MK571 while verapamil had no effect. On the other hand, efflux of Rhodamin 123, a Pgp-glycoprotein substrate, from lung cells reacted to inhibition by verapamil, while MK571 had no effect. Modulation of glutathion content of lung cells by N-acetyl cystein and buthionine sulfoximine shifted CDF efflux toward higher or lower rates, respectively. These experiments confirm that MRP1 function can be followed in the attached cells in vitro under non-toxic concentrations of the substrates without the need to harvest and destroy the cells. Abdelrahman Torky and Ekkehard Stehfest have contributed equally to this study     

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.     

Collateral sensitivity of resistant MRP1-overexpressing cells to flavonoids and derivatives through GSH efflux

The multidrug resistance protein 1 (MRP1) is involved in multidrug resistance of cancer cells by mediating drug efflux out of cells, often in co-transport with glutathione (GSH). GSH efflux mediated by MRP1 can be stimulated by verapamil. In cells overexpressing MRP1, we have previously shown that verapamil induced a huge intracellular GSH depletion which triggered apoptosis of the cells. That phenomenon takes place in the more global anticancer strategy called “collateral sensitivity” and could be exploited to eradicate some chemoresistant cancer cells. Seeking alternative compounds to verapamil, we screened a library of natural flavonoids and synthetic derivatives. A large number of these compounds stimulate MRP1-mediated GSH efflux and the most active ones have been evaluated for their cytotoxic effect on MRP1-overexpressing cells versus parental cells. Interestingly, some are highly and selectively cytotoxic for MRP1-cells, leading them to apoptosis. However, some others do not exhibit any cytotoxicity while promoting a strong GSH efflux, indicating that GSH efflux is necessary but not sufficient for MRP1-cells apoptosis. In support to this hypothesis, structure activity relationships show that the absence of a hydroxyl group at position 3 of the flavonoid C ring is an absolute requirement for induction of MRP1-cells death, but is not for GSH efflux stimulation. Chrysin (compound 8) and its derivatives, compounds 11 and 22, exhibit a high selectivity toward MRP1-cells with a IC₅₀ value of 4.1 μM for compound 11 and 4.9 μM for chrysin and compound 22, making them among the best described selective killer compounds of multidrug ABC transporter-overexpressing cells.     

慢性阻塞性肺疾病与多药耐药相关蛋白1的相关性研究进展

慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)是肺部常见疾病,与肺部对有害气体或有害颗粒的异常炎症反应有关,其中炎症反应和氧化应激等扮演着重要的角色。多药耐药相关蛋白(multidrug resistance-associated protein 1,MRP1)是一种依赖于ATP介导底物跨膜转运蛋白。近年来研究表明,MRP1在肺部广泛表达,且有着特定的生物学功能,如介导外源性有毒物质的外排,内源性物质炎症介质LTC4及GSH等的跨膜转运,其生物学功能的改变在COPD的发生和发展过程中发挥着重要作用。通过了解MRP1在肺部疾病的功能,有利于进一步理解MRP1和COPD的关系,从而更好地指导COPD的治疗。     

Effects of new 4-aryl-1,4-dihydropyridines and 4-arylpyridines on drug efflux mediated by multidrug resistance-associated protein 1

The purpose of this study is to evaluate the effects of newly synthesized 4-aryl-1,4-dihydropyridine and pyridines on drug efflux mediated by multidrug resistance-associated protein 1 (MRP1, ABCC1). These compounds were designed to maximize inhibition of P-glycoprotein and minimize calcium channel binding activity, based on structure modifications of niguldipine. A [3H]vinblastine accumulation study was conducted in human small cell lung cancer H69AR (overexpressing MRP1) and wild type H69 cells. Five out of 16 dihydropyridines and 6 out of 9 pyridines were found to significantly increase the intracellular accumulation of vinblastine in resistant H69AR cells (p<0.01) at a concentration of 2.5 microM. Daunomycin accumulation studies, determined using a flow cytometric assay, were also performed in H69AR and human pancreatic adenocarcinoma Panc-1 cells and the results were highly correlated with those obtained from the [3H]vinblastine accumulation studies. Four compounds, which significantly increased vinblastine accumulation, were tested for their effect on daunomycin cytotoxicity in H69AR cells and found to significantly decrease the IC50 of daunomycin, confirming the accumulation study results. Compounds were also tested for their effect on intracellular glutathione (GSH) concentrations, a cosubstrate for MRP1-mediated efflux in H69AR and Panc-1 cells. No significant changes in the intracellular GSH level were observed in H69AR cells after treatment with these test compounds. However, following a 2-hr and 24-hr incubation with a dihydropyridine compound, Im, and its pyridine derivative IIm, there was a small (approximately 20%) but statistically significant decrease in intracellular GSH in Panc-1 cells. Our results indicate that some dihydropyridine and pyridine compounds in our series could inhibit MRP1-mediated transport and that GSH modulation plays a minor, if any, role in this effect.     

Protection of wheat bran feruloyl oligosaccharides against free radical-induced oxidative damage in normal human erythrocytes

The present work assessed the protective effect of water-soluble feruloyl oligosaccharides (FSH), ferulic acid ester of oligosaccharides from wheat bran, against in vitro oxidative damage of normal human erythrocytes induced by a water-soluble free radical initiator, 2,2′-azobis-2-amidinopropane dihydrochloride (AAPH). In the whole process of AAPH-initiated oxidation, hemolysis occurred quickly after the lag time. The rate of hemolysis is correlated dose-dependently with AAPH concentration. Significant decrease in reduced glutathione (GSH) levels of erythrocyte with concomitant enhancement in oxidized gluthione (GSSG) levels was noticed. It was also observed that lipid and protein peroxidation of erythrocytes induced by AAPH was significantly increased, and scanning electron microscopy observations showed that AAPH induced obvious morphological alteration in the erythrocytes from a smooth discoid to an echinocytic form. FSH suppressed depletion of GSH, lipid peroxidation, and methaemoglobin and protein carbonyl group formation of erythrocytes in concentration- and time-dependent manners, remarkably delayed AAPH-induced hemolysis. Morphological changes to erythrocyte caused by AAPH were effectively protected by FSH. It was also observed that FSH could work synergistically with endogenous antioxidants in erythrocytes. These results indicated that FSH efficiently protected normal human erythrocytes against oxidative stress, and they could be used as a potential source of natural antioxidants.