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To date, 12 members of the human ABCA subfamily are identified. They share a high degree of sequence conservation and have been mostly related with lipid trafficking in a wide range of body locations. Mutations in some of these genes have been described to cause severe hereditary diseases related with lipid transport, such as fatal surfactant deficiency or harlequin ichthyosis. In addition, most of them are hypothesized to participate in the subcellular sequestration of drugs, thereby being responsible for the resistance of several carcinoma cell lines against drug treatment [1, 2].

Subfamily ABCC contains thirteen members and nine of these transporters are referred to as the Multidrug Resistance Proteins (MRPs). The MRP proteins are found throughout nature and they mediate many important functions. They are known to be involved in ion transport, toxin secretion, and signal transduction [7, 2].

The purpose of this minireview is to show that a new paradigm is developing regarding hepatic bile flow. The focus thus far has been on carrier-mediated transport of bile acids and other solutes, such as glutathione, which create an osmotic gradient for the transcellular and paracellular flow of water into canaliculi. In addition to the physicochemical properties of bile acids, which govern the osmotic gradient, data now exist showing that the tight junctions governing paracellular water flow and Aquaporin-8 water channels governing transcellular water flow are regulated independently. Thus, the rate of water flow into the canaliculus in response to bile acid transport is variable and determines canalicular bile acid concentration, which affects the production and solubilization of cholesterol-lecithin vesicles. These new considerations modify thinking regarding the occurrence of cholestasis and its progression and reorient the design of experimental studies that can distinguish the different determinants of bile flow. SIGNIFICANCE STATEMENT The paradigm that water flow into the canaliculus is determined only by the rate of carrier-mediated transport has been challenged recently by the changes that occur in hepatic bile composition in the Claudin-2 knockout mouse and with the cholestatic effect of estradiol 17b-D-glucuronide. Thus, a respective reduction in paracellular or transcellular canalicular water flow, probably via Aquaporin 8, has no significant effect on bile acid excretion. Fil: Marinelli, Raul Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Vore, Mary. University of Kentucky; Estados Unidos Fil: Javitt, Norman B.. University of New York. School of Medicine; Estados Unidos

Doxorubicin (DOX) is a potent chemotherapeutic agent known to cause acute and long-term cognitive impairments in cancer patients. Cognitive function is presumed to be primarily mediated by neuronal circuitry in the frontal cortex (FC) and hippocampus, where glutamate is the primary excitatory neurotransmitter. Mice treated with DOX (25 mg/kg i.p.) were subjected to in vivo recordings under urethane anesthesia at 24h post-DOX injection or 5 consecutive days of cognitive testing (Morris Water Maze; MWM). Using novel glutamate-selective microelectrode arrays, amperometric recordings measured parameters of extracellular glutamate clearance and potassium-evoked release of glutamate within the medial FC and dentate gyrus (DG) of the hippocampus. By 24h post-DOX injection, glutamate uptake was 45% slower in the FC in comparison to saline-treated mice. In the DG, glutamate took 48% longer to clear than saline-treated mice. Glutamate overflow in the FC was similar between treatment groups, however, it was significantly increased in the DG of DOX treated mice. MWM data indicated that a single dose of DOX impaired swim speed without impacting total length traveled. These data indicate that systemic DOX treatment changes glutamate neurotransmission in key nuclei associated with cognitive function within 24h, without a lasting impact on spatial learning and memory. Understanding the functional effects of DOX on glutamate neurotransmission may help us understand and prevent some of the debilitating side effects of chemotherapeutic treatment in cancer survivors.

ATP binding cassette (ABC) transporters are transmembrane proteins expressed in secretory epithelia like the liver, kidneys and intestine, in the epithelia exhibiting barrier function such as the blood-brain barrier and placenta, and to a much lesser extent, in tissues like reproductive organs, lungs, heart and pancreas, among others. They regulate internal distribution of endogenous metabolites and xenobiotics including drugs of therapeutic use and also participate in their elimination from the body. We here describe the function and regulation of ABC transporters in the heart and small intestine, as examples of extrahepatic tissues, in which ABC proteins play clearly different roles. In the heart, they are involved in tissue pathogenesis as well as in protecting this organ against toxic compounds and druginduced oxidative stress. The small intestine is highly exposed to therapeutic drugs taken orally and, consequently, ABC transporters localized on its surface strongly influence drug absorption and pharmacokinetics. Examples of the ABC proteins currently described are Multidrug Resistance-associated Proteins 1 and 2 (MRP1 and 2) for heart and small intestine, respectively, and P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) for both organs. Fil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Zhang, Wei. University of Kentucky; Estados Unidos Fil: Zecchinati, Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Mottino, Aldo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Vore, Mary. University of Kentucky; Estados Unidos

Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0 ng/mL for GSH and 1.0 ng/mL for GSSG with lower limits of detection at 0.5 ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases.

Cardiovascular complications are major side effects of many anticancer drugs. Accumulated evidence indicates that oxidative stress in mitochondria plays an important role in cardiac injury, but how mitochondrial redox mechanisms are involved in cardiac dysfunction remains unclear. Here, we demonstrate that 4-hydroxy-2-nonenal (HNE) activates the translocation of the mitochondrial apoptosis inducing factor (AIFm2) and facilitates apoptosis in heart tissue of mice and humans. Doxorubicin treatments significantly enhance cardiac levels of HNE and AIFm2. HNE adduction of AIFm2 inactivates the NADH oxidoreductase activity of AIFm2 and facilitates its translocation from mitochondria. His 174 on AIFm2 is the critical target of HNE adduction that triggers this functional switch. HNE adduction and translocation of AIFm2 from mitochondria upon Doxorubicin treatment are attenuated by superoxide dismutase mimetics. These results identify a previously unrecognized role of HNE with important consequences for mitochondrial stress signaling, heart failure, and the side effects of cancer therapy.

Doxorubicin (DOX) induces dose-dependent cardiotoxicity in part due to its ability to induce oxidative stress. We showed that loss of multidrug resistance-associated protein 1 (Abcc1/Mrp1) potentiates DOX-induced cardiac dysfunction in mice in vivo. Here, we characterized DOX toxicity in cultured cardiomyocytes (CM) and cardiac fibroblasts (CF) derived from C57BL wild type (WT) and Mrp1 null (Mrp1−/−) neonatal mice. CM accumulated more intracellular DOX relative to CF but this accumulation did not differ between genotypes. Following DOX (0.3–4 μM), Mrp1−/− CM, and CF, especially CM, showed a greater decrease in viability and increased apoptosis and DNA damage, demonstrated by higher caspase 3 cleavage, poly (ADP-ribose) polymerase 1 (PARP) cleavage and phosphorylated histone H2AX (γH2AX) levels versus WT cells. Saline- and DOX-treated Mrp1−/− cells had significantly higher intracellular GSH and GSSG compared with WT cells (P

Cardiotoxicity is a major dose-limiting adverse effect of doxorubicin (DOX), mediated in part by overproduction of reactive oxygen species and oxidative stress. Abcc1 (Mrp1) mediates the efflux of reduced and oxidized glutathione (GSH, GSSG) and is also a major transporter that effluxes the GSH conjugate of 4-hydroxy-2-nonenal (HNE; GS-HNE), a toxic product of lipid peroxidation formed during oxidative stress. To assess the role of Mrp1 in protecting the heart from DOX-induced cardiac injury, wild-type (WT) and Mrp1 null (Mrp1(-/-)) C57BL/6 littermate mice were administered DOX (15 mg/kg) or saline (7.5 ml/kg) i.v., and heart ventricles were examined at 72 hours. Morphometric analysis by electron microscopy revealed extensive injuries in cytosol, mitochondria, and nuclei of DOX-treated mice in both genotypes. Significantly more severely injured nuclei were observed in Mrp1(-/-) versus WT mice (P = 0.031). GSH and the GSH/GSSG ratio were significantly increased in treatment-naive Mrp1(-/-) versus WT mice; GSH remained significantly higher in Mrp1(-/-) versus WT mice after saline and DOX treatment, with no changes in GSSG or GSH/GSSG. GS-HNE, measured by mass spectrometry, was lower in the hearts of treatment-naive Mrp1(-/-) versus WT mice (P < 0.05). DOX treatment decreased GS-HNE in WT but not Mrp1(-/-) mice, so that GS-HNE was modestly but significantly higher in Mrp1(-/-) versus WT hearts after DOX. Expression of enzymes mediating GSH synthesis and antioxidant proteins did not differ between genotypes. Thus, despite elevated GSH levels in Mrp1(-/-) hearts, DOX induced significantly more injury in the nuclei of Mrp1(-/-) versus WT hearts.

Doxorubicin (DOX), one of the most effective anticancer drugs, is known to generate progressive cardiac damage, which is due, in part, to DOX-induced reactive oxygen species (ROS). The elevated ROS often induce oxidative protein modifications that result in alteration of protein functions. This study demonstrates that the level of proteins adducted by 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, is significantly increased in mouse heart mitochondria following DOX treatment. A redox proteomics method involving 2D electrophoresis followed by mass spectrometry and investigation of protein data bases identified several HNE-modified mitochondria proteins, which were verified by HNE-specific immunoprecipitation in cardiac mitochondria from the DOX-treated mice. The majority of the identified proteins are related to mitochondrial energy metabolism. These include proteins in the citric acid cycle (TCA) and electron transport chain (ETC). The enzymatic activities of the HNE-adducted proteins were significantly reduced in DOX-treated mice. Consistent with the decline in the function of the HNE adducted proteins, the respiratory function of cardiac mitochondria as determined by oxygen consumption rate (OCR) was also significantly reduced after DOX treatment. The treatment with Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnP, an SOD mimic, averted the doxorubicin-induced mitochondrial dysfunctions as well as the HNE protein adductions. Together, the results demonstrate that free radical-mediated alteration of energy metabolism is an important mechanism mediating DOX-induced cardiac injury suggesting that metabolic intervention may represent a novel approach to preventing cardiac injury after chemotherapy.

ABC transporters including MRP2, MDR1 and BCRP play a major role in tissue defense. Epidemiological and experimental studies suggest a cytoprotective role of estrogens in intestine,though the mechanism remains poorly understood. We evaluated whether pharmacologic concentrations of ethynylestradiol (EE, 0.05 pM to 5 nM), or concentrations of genistein (GNT)associated with soy ingestion (0.1 to 10 µM), affect the expression and activity of multidrug resistance proteins MRP2, MDR1 and BCRP using Caco-2 cells, an in vitro model of intestinal epithelium. We found that incubation with 5 pM EE and 1 µM GNT for 48 h increased expression and activity of both MRP2 and MDR1. Estrogens did not affect expression of BCRP protein at any concentration studied. Irrespective of the estrogen tested, up-regulation of MDR1 and MRP2 protein was accompanied by increased levels of MDR1 mRNA, whereas MRP2 mRNA remained unchanged. Cytotoxicity assays demonstrated association of MRP2 and MDR1 up-regulation with increased resistance to cell death induced by 1-chloro-2,4-dinitrobenzene, an MRP2 substrate precursor, and by paraquat, an MDR1 substrate. Experiments using an estrogen receptor (ER) antagonist implicate ER participation in MRP2 and MDR1 regulation. GNT but not EE increased the expression of ERβ, the most abundant form in human intestine and in Caco-2 cells, which could lead in turn to increased sensitivity to estrogens. We conclude that specific concentrations of estrogens can confer resistance against cytotoxicity in Caco-2 cells, due in part to positive modulation of ABC transporters involved in extrusion of their toxic substrates. Although extrapolation of these results to the in vivo situation must be cautiously done, the data could explain tentatively the cytoprotective role of estrogens against chemical injury in intestine. Fil: Arias, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Rigalli, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Perdomo, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Vore, Mary. University Of Kentucky; Estados Unidos Fil: Catania, Viviana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Mottino, Aldo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina

ASPET has published The Journal of Pharmacology and Experimental Therapeutics since 1909. Pharmacological Reviews began in 1949, followed by Molecular Pharmacology in 1965 and Drug Metabolism and Disposition in 1973. Pharmacology Research & Perspectives , jointly published with the British

A group of European national research funding organizations, with the support of the European Commission and the European Research Council, have announced the launch of cOAlition S (“European funders seek to end reign of paywalled journals,” M. Enserink, In Depth, 7 September 2018, p. [957][1]). According to the plan, after 1 January 2020, scientific publications reporting the results of publicly funded research must be published in compliant open-access journals or on open-access platforms. However, the requirement to publish in an open-access journal does not consider the most important aspect of publishing: selecting a journal that has a strong record of rigorous and high-quality review. This is essential to ensuring that the science is credible. Journal quality is built on a strong track record of publishing significant and impactful manuscripts in a given field. The current Plan S emphasizes only the open-access aspect of the journal, not the quality of the science the journal publishes. For over a century, academic societies have developed scientific journals that provide rigorous scientific review of submitted manuscripts. To do so, societies must recruit leadership (such as editors and editorial board members) and provide fiduciary oversight for journals. These responsibilities require highly trained personnel and are expensive. In turn, these journals provide society members with a venue for publishing their research and advancing the discipline. For societies that self-publish, the proceeds from the journals fund activities such as scientific meetings, which focus on the presentation of current research and exchange of information, and mentoring and financial support of young scientists, which are essential to sustaining a rich scientific community. As members of the International Union of Basic and Clinical Pharmacology, the worldwide body for pharmacological societies, we believe using only open-access journals will negatively affect those activities of our professional societies. [1]: http://www.sciencemag.org/content/361/6406/957

Objective Doxorubicin-induced acute cardiotoxicity is associated with the Gly671Val (G671V; rs45511401) variant of multidrug resistance-associated protein 1 (MRP1). Doxorubicin redox cycling causes lipid peroxidation and generation of the reactive electrophile, 4-hydroxy-2-trans-nonenal (HNE). Glutathione forms conjugates with HNE, yielding an MRP1 substrate, GS-HNE, whose intracellular accumulation can cause toxicity. Methods We established stable HEK293 cell lines overexpressing wild-type MRP1 (HEKMRP1), G671V (HEKG671V), and R433S (HEKR433S), a variant not associated with doxorubicin-induced cardiotoxicity and investigated the sensitivity of HEKG671V cells to doxorubicin and transport capacity of G671V toward GS-HNE. Results In ATP-dependent transport studies using plasma membrane-derived vesicles, the Vmax (pmol/min/mg) for GS-HNE transport was the lowest for G671V (69±4) and the highest for R433S (972±213) compared with wild-type MRP1 (416±22), whereas the Km values were 2.8±0.4, 6.0 or more, and 1.7±0.2 µmol/l, respectively. In cells, the doxorubicin IC50 (48 h) was not different in HEKMRP1 (463 nmol/l) versus HEKR433S (645 nmol/l), but this parameter was significantly lower in HEKG671V (181 nmol/l). HEKG671V retained significantly (approximately 20%) more, whereas HEKR433S retained significantly less intracellular doxorubicin than HEKMRP1. Similarly, HEKG671V cells treated with 1.5 µmol/l of doxorubicin for 24 h retained significantly more GS-HNE. In cells treated with 0.5 µmol/l of doxorubicin for 48 , glutathione and glutathione disulfide levels and the glutathione/glutathione disulfide ratio were significantly decreased in HEKG671V versus HEKMRP1; these values were similar in HEKR433S versus HEKMRP1. Conclusion These data suggest that decreased MRP1-dependent GS-HNE efflux contributes to increased doxorubicin toxicity in HEKG671V and potentially in individuals carrying the G671V variant.

Multidrug resistance-associated protein 2 (MRP2; ABCC2) mediates the biliary excretion of glutathione, glucuronide, and sulfate conjugates of endobiotics and xenobiotics. Single nucleotide polymorphisms (SNPs) of MRP2 contribute to interindividual variability in drug disposition and ultimately in drug response.To characterize the transport function of human wild-type (WT) MRP2 and four SNP variants, S789F, A1450T, V417I, and T1477M.The four SNP variants were expressed in Sf9 cells using recombinant baculovirus infection. The kinetic parameters [Km, (μmol/l); V(max), (pmol/mg/min); the Hill coefficient] of ATP-dependent transport of leukotriene C(4) (LTC(4)), estradiol-3-glucuronide (E(2)3G), estradiol-17β-glucuronide (E(2)17G), and tauroursodeoxycholic acid (TUDC) were determined in Sf9-derived plasma membrane vesicles. Transport activity was normalized for expression level.The V(max) for transport activity was decreased for all substrates for S789F, and for all substrates except E(2)17G for A1450T. V417I showed decreased apparent affinity for LTC(4), E(2)3G, and E(2)17G, whereas transport was similar between wild-type (WT) and T1477M, except for a modest increase in TUDC transport. Examination of substrate-stimulated MRP2-dependent ATPase activity of S789F and A1450T, SNPs located in MRP2 nucleotide-binding domains (NBDs), demonstrated significantly decreased ATPase activity and only modestly decreased affinity for ATP compared with WT.SNPs in the NBDs (S789F in the D-loop of NBD1, or A1450T near the ABC signature motif of NBD2) variably decreased the transport of all substrates. V417I in membrane spanning domain 1 selectively decreased the apparent affinity for the glutathione and glucuronide conjugated substrates, whereas the T1477M SNP in the carboxyl terminus altered only TUDC transport.

Tetrahydroxy bile acids become major biliary bile acids in Bsep(−/−) mice and Fxr(−/−) mice fed cholic acid; we characterized disposition of these novel bile acids that also occur in patients with cholestasis. We investigated mouse Mrp2 (mMrp2) and P-glycoprotein [(P-gp) mMdr1a]-mediated transport of a tetrahydroxy bile acid, 6α-OH-taurocholic acid (6α-OH-TC), and its biliary excretion in wild-type and Mrp2(−/−) mice in the presence or absence of N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), a P-gp and breast cancer resistance protein inhibitor. 6α-OH-TC was rapidly excreted into bile of wild-type mice (78% recovery); coinfusion of GF120918 had no significant effect. In Mrp2(−/−) mice, biliary excretion was decreased (52% recovery) and coinfusion of GF120918 further decreased these values (34% recovery). In wild-type, but not Mrp2(−/−), mice, 6α-OH-TC increased bile flow 2.5-fold. Membrane vesicle transport studies of 6α-OH-TC (0.05–0.75 mM) yielded saturation kinetics with a higher apparent affinity for mMrp2 (Km = 0.13 mM) than for mMdr1a (Km = 0.33 mM); mBsep transported 6α-OH-TC with positive cooperativity (Hill slope = 2.1). Human multidrug resistance-associated protein (MRP) 2 and P-gp also transported 6α-OH-TC but with positive cooperativity (Hill slope = 3.6 and 1.6, respectively). After intraileal administration, the time course of 6α-OH-TC biliary recovery was similar to that of coinfused taurocholate, implying that 6α-OH-TC can undergo enterohepatic cycling. Thus, Mrp2 plays a key role in 6α-OH-TC biliary excretion, whereas P-glycoprotein plays a secondary role; Bsep likely mediates excretion of 6α-OH-TC in the absence of Mrp2 and P-gp. In Bsep(−/−) mice, efficient synthesis of tetrahydroxy bile acids that are Mrp2 and P-gp substrates can explain the noncholestatic phenotype.

Lactation is associated with increased expression of bile acid transporters and an increased size and hydrophobicity of the bile acid pool in rats. ATP-binding cassette (ABC) transporters multidrug resistance protein 2 (Mdr2), Abcb11 [bile salt export pump (Bsep)], and Abcg5/Abcg8 heterodimers are essential for the biliary secretion of phospholipids, bile acids, and cholesterol, respectively. We investigated the expression of these transporters and secretion of their substrates in female control and lactating Sprague Dawley rats and C57BL/6 mice. Expression of Abcg5/Abcg8 mRNA was decreased by 97 and 60% by midlactation in rats and mice, respectively; protein levels of Abcg8 were below detection limits in lactating rats. Mdr2 mRNA expression was decreased in lactating rats and mice by 47 and 59%, respectively. Despite these changes in transporter expression, basal concentrations of cholesterol and phospholipid in bile were unchanged in rats and mice, whereas increased Bsep mRNA expression in early lactation coincided with an increased basal biliary bile acid concentration in lactating mice. Following taurocholate infusion, coupling of phospholipid and taurocholate secretion in bile of lactating mice was significantly impaired relative to control mice, with no significant changes in maximal secretion of cholesterol or bile acids. In rats, taurocholate infusion revealed a significantly impaired coupling of cholesterol to taurocholate secretion in bile in lactating vs. control animals. These data reveal marked utilization of an Abcg5/Abcg8-independent mechanism for basal biliary cholesterol secretion in rats during lactation, but a dependence on Abcg5/g8 for maximal biliary cholesterol secretion.

MRP2 (ABCC2), a member of the ATP binding cassette superfamily of efflux transporters that mediates the apical efflux of organic anions from hepatocytes, enterocytes, and renal epithelial cells, is postulated to undergo post-transcriptional regulation. The MRP2 5′-untranslated region (5′UTR) contains seven upstream start codons and six upstream open reading frames (uORFs). Ribonuclease protection assays in human liver, placenta, kidney, small intestine, and HepG2 cells identified multiple MRP2 transcription initiation sites. We investigated MRP2 5′UTRs [−247 (−247 to −1), −204 (−204 to −1), or −99 (−99 to −1)] for their effects on regulation of gene expression with the use of transient gene expression in HepG2 cells and in vitro translation assays. In HepG2 cells transfected with SV40-MRP2-5′UTR-Luciferase cassettes, luciferase activities of constructs −247 and −204 were significantly lower than that of −99. Disruption of the uORFs at −105 and −74 nucleotides by mutation of ATGs to AAG enhanced luciferase activity significantly without affecting luciferase mRNA expression. The translation efficiencies of T7-5′UTR-Luciferase cassettes determined in vitro were consistent with transfected HepG2 cells and showed that inhibition of translation by the −105 uORF occurred only in the cis configuration and not in the trans configuration and that inhibition of translation by the −105 uORF was independent of the encoded peptide sequence. Characterization of an MRP2 polymorphism, −24C>T, in the MRP2 5′UTR, demonstrated no effect on mRNA expression or downstream ORF translation. These data indicate for the first time that the 5′UTR of MRP2 mRNA transcripts and the uORF at −105 markedly influence MRP2 translation.

The effect of the cholestatic estrogens ethynylestradiol (EE) and estradiol 17beta-D-glucuronide (E2-17G) on expression and activity of intestinal multidrug resistant-associated protein 2 (Mrp2, Abcc2) was studied in rats. Expression and localization of Mrp2 were evaluated by Western blotting, real-time polymerase chain reaction, and confocal immunofluorescence microscopy. Mrp2 transport activity toward dinitrophenyl-S-glutathione (DNP-SG) was assessed in vitro in intestinal sacs. EE, administered subcutaneously at a 5 mg/kg b.wt. dose, for 5 consecutive days, produced a marked decrease in Mrp2 expression at post-transcriptional level, without affecting its normal localization at the apical membrane of the enterocyte. This effect was selective because expression of other ATP-binding cassette proteins such as breast cancer resistance protein and Mrp3 were not affected and that of multidrug resistance protein 1 was only minimally impaired. Consistent with down-regulation of expression of Mrp2, a significant impairment in serosal to mucosal transport of DNP-SG and in protection against absorption of this same compound were registered. Simultaneous administration of EE with spironolactone (200 micromol/kg b.wt./day for 3 days), an Mrp2 inducer, prevented these alterations, confirming down-regulation of expression of Mrp2 by EE as a major component of functional changes. Incorporation of E2-17G (30 microM) in the serosal medium of intestinal sacs decreased serosal to mucosal transport of DNP-SG, probably because of competitive inhibition, without affecting normal Mrp2 expression or localization. Our data indicate impairment of function of intestinal Mrp2 by both cholestatic estrogens, although through a different mechanism. This finding represents an aggravation of deteriorated hepatic Mrp2 function that could further increase bioavailability of specific xenobiotics after oral exposure.

Multidrug resistance-associated protein 1 (Mrp1; Abcc1) is expressed in sarcolemma of murine heart, where it probably protects the cardiomyocyte by mediating efflux of endo- and xenobiotics. We used doxorubicin (DOX), a chemotherapeutic drug known to induce oxidative stress and thereby cardiac injury, as a model cardiotoxic compound and observed changes in the Mrp1 expression pattern in cardiac tissue of DOX-versus saline-treated mice. Confocal immunofluorescent and immunogold electron microscopy, together with subcellular fractionation followed by immunoblot analyses and transport measurements, localized functional Mrp1 to mitochondria after DOX. Expressions of Mrp1 in heart homogenate, sarcolemma, and submitochondrial particles (SMP) were increased 1.6-, 2-, and 3-fold, respectively, at 24 h after DOX. Mitochondrial Mrp1 expression was markedly increased 72 h after DOX, whereas transport of Mrp1 substrates in SMP was maximal at 24 h. ATP-dependent transport in SMP occurred into an osmotically sensitive space and was inhibited by the anti-MRP1 antibody QCRL3. Adduction of a 190-kDa protein with the reactive lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was detected in SMP and was maximal at 72 h after DOX; immunoprecipitation confirmed Mrp1-HNE adduction. In vitro, HNE (10 μM) inhibited mitochondrial respiration and transport activity in SMP, suggesting that Mrp1 is adversely affected by oxidative stress. These data demonstrate that after DOX, functional Mrp1 is detected in mitochondria in addition to that in sarcolemma; however, adduction with HNE inhibits Mrp1 activity. Mrp1 may serve to protect the heart by mediating the efflux of toxic products of oxidative stress from mitochondria and cardiomyocytes.

Bile formation represents a key liver function by which xenobiotics and endogenous metabolites such as cholesterol, bilirubin, and hormones are eliminated from the body (1, 2). Efflux of solutes by ATP-dependent transporters at the canalicular membrane of hepatocytes provide the driving force for osmotic bile formation; among these transporters, the bile salt export pump (Bsep; Abcc11) and the multidrug resistance-associated protein 2 (Mrp2; Abcc2), responsible for transporting bile salts and glutathione (GSH) and glucuronide conjugates, respectively, play a central role in this process (1, 3). Alterations of canalicular transporter expression, localization, or activity can lead to cholestasis (3). Short-term endocytic retrieval of Bsep and Mrp2 occurs in several experimental models of cholestasis (3, 4). This mechanism likely impairs secretory function by reducing the total number of efflux transporters in the canalicular domain and therefore their transport capacity (Vmax) within minutes. Stimulation of exocytic insertion of transporter-containing vesicles by administration of tauroursodeoxycholate or cyclic AMP prevents cholestasis, thus supporting this hypothesis (5). The endogenous estradiol metabolite, estradiol 17β-D-glucuronide (E217G) is a useful model to study the manifestations associated with estrogen-induced cholestasis. Indeed, E217G induces a dose-dependent, acute and reversible cholestasis by impairing both Bsep-mediated efflux of bile acids and the Mrp2-mediated efflux of GSH (6). As a likely mechanism, E217G causes a microtubule-independent, endocytic retrieval of both Bsep and Mrp2 (7, 8); spontaneous exocytic re-insertion of these canalicular transporters in a microtubule-dependent fashion is linked to restoration of bile flow and canalicular transport activity (9). Recent evidence indicates that activation of protein kinase C (PKC) is associated with impairment of the secretory function of hepatocytes, and ultimately leads to cholestasis (10–12). Thymeleatoxin-mediated selective activation of the classical Ca2+-dependent PKC isoforms (cPKC), of which PKCα and βII are expressed in hepatocytes (13), is cholestatic in perfused rat liver (PRL) (14). Similarly, oxidative stress-induced impairment of bile salt secretory function in isolated rat hepatocyte couplets (IRHC) is dependent on activation of PKCα (15). Interestingly, this PKCα activation is also associated with retrieval of Bsep and the associated loss of its secretory function (14, 15). In the present study, we determined whether cPKC isoforms mediate the cholestasis induced by E217G in the in situ PRL and in IRHC. Our results unambiguously demonstrate that cPKC isoforms are critical to the impairment of localization and function of Bsep and Mrp2 induced by E217G.

Estradiol-17β-d-glucuronide (E217G) induces immediate and profound but transient cholestasis in rats when administered as a single bolus dose. Here, we examined the consequence of sustained E217G cholestasis and assessed the function and localization of the tight junctional proteins zonula occludens-1 (ZO-1) and occludin and of the canalicular transporter multidrug resistance-associated protein-2 (Mrp2). An initial dose of E217G (15 μmol/kg iv) followed by five subsequent doses of 7.5 μmol/kg from 60 to 240 min induced a sustained 40–70% decrease in bile flow. Following their biliary retrograde administration, cholera toxin B subunit-FITC or horseradish peroxidase were detected at the sinusoidal domain, indicating opening of the paracellular route; this occurred as early as 15 min after the first dose as well as 15 min after the last dose of E217G, but not following the administration of vehicle in controls. Localization of ZO-1 and occludin was only slightly affected under acute cholestatic conditions but was severely disrupted under sustained cholestasis, with their appearance suggesting a fragmented structure. Endocytic internalization of Mrp2 to the pericanalicular region was apparent 20 min after a single E217G administration; however, Mrp2 was found more deeply internalized and partially redistributed to the basolateral membrane under sustained cholestasis. In conclusion, acute E217G-induced cholestasis increased permeability of the tight junction, while sustained cholestasis provoked a significant redistribution of ZO-1, occludin, and Mrp2 in addition to increased permeability of the tight junction. Altered tight junction integrity likely contributes to impaired bile secretion and may be causally related to changes in Mrp2 localization.

Injury to nontargeted tissues in chemotherapy often complicates cancer treatment by limiting therapeutic dosages of anticancer drugs and by impairing the quality of life of patients during and after treatment. Oxidative stress, directly or indirectly caused by chemotherapeutics as exemplified by doxorubicin, is one of the underlying mechanisms of the toxicity of anticancer drugs in noncancerous tissues, including the heart and brain. A comprehensive understanding of the mechanisms of oxidative injury to normal tissue will be essential for the improvement of strategies to prevent or attenuate the toxicity of chemotherapeutic agents without compromising their chemotherapeutic value.

Adriamycin (ADR), a potent anti-tumor agent, produces reactive oxygen species (ROS) in cardiac tissue. Treatment with ADR is dose-limited by cardiotoxicity. However, the effect of ADR in the other tissues, including the brain, is unclear because ADR does not pass the blood-brain barrier. Some cancer patients receiving ADR treatment develop a transient memory loss, inability to handle complex tasks etc., often referred to by patients as chemobrain. We previously demonstrated that ADR causes CNS toxicity, in part, via systemic release of cytokines and subsequent generation of reactive oxygen and nitrogen species (RONS) in the brain. Here, we demonstrate that treatment with ADR led to an increased circulating level of tumor necrosis factor-alpha in wild-type mice and in mice deficient in the inducible form of nitric oxide (iNOSKO). However, the decline in mitochondrial respiration and mitochondrial protein nitration after ADR treatment was observed only in wild-type mice, not in the iNOSKO mice. Importantly, the activity of a major mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD), was reduced and the protein was nitrated. Together, these results suggest that NO is an important mediator, coupling the effect of ADR with cytokine production and subsequent activation of iNOS expression. We also identified the mitochondrion as an important target of ADR-induced NO-mediated CNS injury.

Oxidative stress in heart and brain by the cancer chemotherapeutic drug adriamycin (ADR), used for treating solid tumors, is well established. Long-term treatment with ADR in breast cancer patients has led to symptoms of cardiomyopathy. Less well recognized, but increasingly well documented, is cognitive dysfunction. After chemotherapy, free radical-mediated oxidative stress has been reported in both heart and brain. We recently showed a significant increase in protein oxidation and lipid peroxidation in brain isolated from mice injected intraperitonially (i.p) with ADR. Systemic administration of ADR also induces tumor necrosis factor-alpha (TNF-alpha), which leads to production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in brain. Circulating TNF also causes mitochondrial dysfunction, leading to apoptotic pathways in brain. Inducible nitric oxide synthase also plays a role in ADR-induced TNF-mediated neurotoxicity. In addition, we previously showed a significant decrease in glutathione (GSH) levels in brain isolated from ADR injected mice, along with increased expression of multidrug-resistant protein-1 (MRP-1), glutathione-S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GR). There was a significant decrease in activity of brain GST. The present study was designed to test the hypothesis that, by elevating brain levels of GSH, the brain would be protected against oxidative stress in ADR-injected mice. gamma-Glutamyl cysteine ethyl ester (GCEE), a precursor of glutathione, injected i.p. (150 mg/ kg body weight) 4 hr prior ADR injection (20 mg/kg body weight) led to significantly decreased protein oxidation and lipid peroxidation in subsequently isolated mice brain compared with brain isolated from ADR-injected mice without GCEE. The GSH levels were restored to the level of brain isolated from saline-injected mice. Furthermore, the enzyme activity of GST was increased in brain isolated from ADR-injected mice previously injected with GCEE compared with the brain isolated from ADR-injected mice previously injected with saline. These results are discussed with regard to potential pharmacological prevention of brain cognitive dysfunction in patients receiving ADR chemotherapy.

Doxorubicin (DOX), an effective cancer chemotherapeutic agent, induces dose-dependent cardiotoxicity, in part due to its ability to cause oxidative stress. We investigated the role of multidrug resistance-associated protein 1 (Mrp1/Abcc1) in DOX-induced cardiotoxicity in C57BL wild-type (WT) mice and their Mrp1 null (Mrp1(-/-)) littermates. Male mice were administered intraperitoneal DOX (3 or 2 mg/kg body weight) or saline twice a week for 3 weeks and examined 2 weeks after the last dose (protocol A total dose: 18 mg/kg) or for 5 weeks, and mice were examined 48 hours and 2 weeks after the last dose (protocol B total dose: 20 mg/kg). Chronic DOX induced body weight loss and hemotoxicity, adverse effects significantly exacerbated in Mrp1(-/-) versus WT mice. In the heart, significantly higher basal levels of glutathione (1.41-fold ± 0.27-fold) and glutathione disulfide (1.35-fold ± 0.16-fold) were detected in Mrp1(-/-) versus WT mice, and there were comparable decreases in the glutathione/glutathione disulfide ratio in WT and Mrp1(-/-) mice after DOX administration. Surprisingly, DOX induced comparable increases in 4-hydroxynonenal glutathione conjugate concentration in hearts from WT and Mrp1(-/-) mice. However, more DOX-induced apoptosis was detected in Mrp1(-/-) versus WT hearts (P < 0.05) (protocol A), and cardiac function, assessed by measurement of fractional shortening and ejection fraction with echocardiography, was significantly decreased by DOX in Mrp1(-/-) versus WT mice (P < 0.05; 95% confidence intervals of 20.0%-24.3% versus 23.7%-29.5% for fractional shortening, and 41.5%-48.4% versus 47.7%-56.7% for ejection fraction; protocol B). Together, these data indicate that Mrp1 protects the mouse heart against chronic DOX-induced cardiotoxicity.

The multidrug resistance protein 2 (MRP2/ABCC2) mediates the biliary excretion of glucuronide and glutathione conjugates of endogenous and exogenous compounds. We examined the activation of human MRP2-mediated ATP-dependent transport by the choleretic bile salt ursodeoxycholic acid (UDC) and its taurine and glycine amidates in Sf9 cell membranes expressing MRP2 using beta-estradiol 17-(beta-D-glucuronide) (E(2)17G) and beta-estradiol 3-(beta-D-glucuronide) (E(2)3G) as substrates. MRP2 transported E(2)3G via classic Michaelis-Menten kinetics (K(m) = 122 microM; V(max) = 3.0 nmol/mg/min), whereas E(2)17G transport showed positive cooperativity (Hill slope, 2.15; K(m) = 75 microM; V(max) = 3.8 nmol/mg/min). UDC, tauroursodeoxycholate, and glycoursodeoxycholate (80-100 microM) maximally stimulated E(2)3G transport 9-, 7.9-, and 3.6-fold, respectively, whereas higher concentrations (1-2 mM) inhibited transport. At low (0.3 microM) concentrations, tauroursodeoxycholate was transported only in the presence of E(2)17G or E(2)3G, but not other MRP2 substrates such as methotrexate, leukotriene C(4), or S-methylglutathione. Kinetic analysis of higher concentrations of tauroursodeoxycholate transport by MRP2 showed positive cooperativity (Hill slope, 1.84; K(m) = 127 microM; V(max) = 779 pmol/mg/min). Taurocholate (2-100 microM) was not detectably transported by MRP2 either alone or in the presence of E(2)17G but was transported in the presence of E(2)3G. Thus, UDC, tauroursodeoxycholate, and glycoursodeoxycholate activated MRP2 transport. Tauroursodeoxycholate was transported by MRP2 and demonstrated positive cooperativity, identifying it as the second MRP2 substrate able to stimulate its own transport. The data suggest MRP2 binding sites that can require specific complementarities between substrates and modulators of MRP2-mediated transport.

Adriamycin (ADR) is a potent anticancer drug, but its use is limited by a dose-dependent cardiotoxicity. Oxidative stress is regarded as the mediating mechanism of ADR cardiotoxicity. However, cardiac proteins that are oxidatively modified have not been well characterized. We took a redox proteomics approach to identify increasingly oxidized murine cardiac proteins after a single injection of ADR (ip, 20 mg/kg body wt). The specific carbonyl levels of three proteins were significantly increased, and these proteins were identified as triose phosphate isomerase (TPI), β-enolase, and electron transfer flavoprotein–ubiquinone oxidoreductase (ETF-QO). TPI and enolase are key enzymes in the glycolytic pathway, and ETF-QO serves as the transporter for electrons derived from a variety of oxidative processes to the mitochondria respiratory chain. Cardiac enolase activity in ADR-treated mice was reduced by 25%, whereas the cardiac TPI activity remained unchanged. Oxidation of purified enolase or TPI via Fenton chemistry led to a 17 or 23% loss of activity, respectively, confirming that a loss of activity was the consequence of oxidation. The observation that these cardiac enzymes involved in energy production are more oxidized resulting from ADR treatment indicates that the bioenergetic pathway is an important target in ADR-initiated oxidative stress.

Multidrug resistance-associated protein 2 (Mrp2/Abcc2), an organic anion transporter present in the apical membrane of hepatocytes, renal epithelial cells, and enterocytes, is postulated to undergo translational regulation. Transcription of rat hepatic Mrp2 mRNA is initiated at multiple sites (-213, -163, -132, and -98 nucleotides relative to the Mrp2 ATG) and contains potential upstream open reading frames (uORFs) in the 5' untranslated region (UTR) starting at -213, -149 and -109 nucleotides. Ribonuclease protection assays demonstrated that transcription of the Mrp2 gene at the various initiation sites was tissue-specific, with the major initiation site in the liver and kidney being -98 and -132 nucleotides, respectively. In the jejunum, the primary and secondary initiation sites were -98 and -132 nucleotides, respectively, with the converse true in the ileum. The relative abundance of these Mrp2 transcripts expressed in tissues varied with age from birth to the adult. HepG2 transient expression assays and in vitro translation assays in which the 5'UTRs were fused with a luciferase reporter showed that the 5'UTR without any uORF (-98 nucleotide) expressed maximal luciferase activity compared with those with one (-132 nucleotides), two (-163 nucleotides), or three (-213 nucleotides) uORFs. Disruption of the uORF by site-directed mutagenesis at nucleotide -109 enhanced luciferase activity 2- to 3-fold, whereas disruption of the uORF at nucleotide -149 had little effect. We conclude that among the uORFs in the Mrp2 5'UTR, the uORF starting at nucleotide -109 probably plays an important role in the regulation of Mrp2 protein expression.

We evaluated the effect of ethynylestradiol (EE) administration (5 mg/kg b.wt. s.c., for 5 consecutive days) on the expression and activity of multidrug resistance-associated protein 3 (Mrp3) in rats. Western blotting analysis revealed decreased Mrp2 (-41%) and increased Mrp3 (+200%) expression by EE. To determine the functional impact of up-regulation of Mrp3 versus Mrp2, we measured the excretion of acetaminophen glucuronide (APAP-glu), a common substrate for both transporters, into bile and perfusate in the recirculating isolated perfused liver (IPL) model. APAP-glu was generated endogenously from acetaminophen (APAP), which was administered as a tracer dose (2 micromol/ml) into the perfusate. Biliary excretion of APAP-glu after 60 min of perfusion was reduced in EE-treated rats (-80%). In contrast, excretion into the perfusate was increased by EE (+45%). Liver content of APAP-glu at the end of the experiment was reduced by 36% in the EE group. The total amount of glucuronide remained the same in both groups. Taken together, these results indicate that up-regulation of Mrp3 led to an exacerbated basolateral versus canalicular excretion of conjugated APAP in IPL. We conclude that induced expression of basolateral Mrp3 by EE may represent a compensatory mechanism to prevent intracellular accumulation of common Mrp substrates, either endogenous or exogenous, due to reduced expression and activity of apical Mrp2.

Multidrug resistance-associated protein 2 (Mrp2, Abcc2), an organic anion transporter present in the apical membrane of hepatocytes, renal epithelial cells, and enterocytes, is postulated to undergo post-transcriptional regulation. We hypothesized that Mrp2 protein undergoes altered rates of protein synthesis or degradation consistent with different Mrp2 protein expression. We analyzed Mrp2 synthesis, expression, and degradation in control female, 19- and 20-day pregnant, and pregnenolone-16alpha-carbonitrile (PCN)-treated rats using in vivo metabolic-labeling studies with [35S]cysteine/methionine or [14C]NaHCO3, polysomal distribution analyses and ribonuclease protection assays (RPA). Mrp2 protein was significantly increased in rats treated with PCN for 2 days but significantly decreased in 19-day pregnant rats relative to controls; no significant differences were observed in Mrp2 mRNA expression among these groups. The measured half-lives of 14C-labeled Mrp2 in control, pregnant, and PCN-treated rats were 27, 36, and 22 h, respectively, and were not significantly different. The rate of incorporation of 35S into Mrp2 was highest in PCN-treated rats. Polysomal distribution analysis of Mrp2 mRNA was consistent with increased Mrp2 protein synthesis after PCN treatment. The major transcription-initiation site for rat liver determined by RPA was -98 nucleotides (nt), with other start sites observed at -213, -163, -132, and -71 nt; use of transcription sites did not differ among the groups. Differences in the degradation of Mrp2 protein cannot explain the post-transcriptional regulation of Mrp2 in control, pregnant, and PCN-treated rats. Rather, the observed difference in protein synthesis suggests an intrinsic role for the translational regulation of rat Mrp2 protein.

Organic anion transporting polypeptides (Oatp) mediate the transport of a wide variety of amphipathic organic substrates. Rat Oatp1b2 and human OATP1B3 are members of a liver-specific subfamily of Oatps/OATPs. We investigated whether prolactin (PRL) and growth hormone (GH) regulated Oatp1b2 and OATP1B3 gene expression via signal transducers and activators of transcription 5 (Stat5). Binding sites for Stat5 transcription factors were located in the promoters of Oatp1b2 and OATP1B3 at -209 to -201 (5'-TTCTGGGAA-3') and -170 to -162 (5'-TTCTGAGAA-3'), respectively. In primary hepatocytes from female and male rats treated with PRL or GH, Oatp1b2 mRNA measured by real-time polymerase chain reaction was significantly induced 2-fold. HepG2 cells were transiently transfected with expression vectors containing Oatp1b2 or OATP1B3 promoter fragments, cDNAs for Stat5a, and the receptors for PRL (PRLR(L)) or GH (GHR), and treated with PRL or GH. PRL and GH induction of Oatp1b2 and OATP1B3 promoter activity required cotransfection of Stat5a and PRLR(L) or GHR. Mutation of the Stat5 binding site in both promoters eliminated hormonal induction. In DNA binding assays, HepG2 cells transfected with cDNAs for Stat5a and PRLR(L) were treated with PRL, and nuclear extracts were probed with a (32)P-labeled oligomer corresponding to -177 to -157 of the OATP1B3 promoter. PRL enhanced the binding of Stat5a to the OATP1B3 promoter and DNA-protein binding was inhibited in competition assays by excess OATP1B3 and Stat5 consensus oligomers but not by mutant Stat5 oligomers. These findings indicate that PRL and GH can regulate Oatp1b2 and OATP1B3 gene expression via the Stat5 signal-transduction pathway.

The mechanisms involved in spironolactone (SL, 200 micromol/kg body weight, 3 days i.p.)-induced choleresis were explored in vivo by evaluating bile salt export pump (Bsep)-, multidrug resistance-associated protein 2 (Mrp2)-, and anion exchanger 2 (AE2)-mediated secretory processes in rat liver. Hepatic bile salt metabolism was also analyzed. Total bile flow was significantly increased by SL, primarily due to an increase in bile salt-independent bile flow, whereas bile salt secretion was decreased. SL did not produce any choleresis in TR(-) rats. SL decreased the de novo bile salt synthesis rate in concordance with impaired microsomal cholesterol 7 alpha-hydroxylase activity, thus leading to a decrease in endogenous bile salt pool size. In contrast, the maximum secretory rate of tauroursodeoxycholate as well as expression of Bsep protein detected by Western blotting were not affected. Thus, decreased bile salt availability for canalicular transport rather than transport capability itself likely explains reduced biliary secretion of bile salts. Biliary secretion of glutathione, an endogenous substrate of Mrp2, and HCO(3)(-), the AE2 substrate, were increased by SL, as a main factor explaining enhanced bile salt-independent bile flow. Western blot studies revealed increased expression of Mrp2 in response to SL whereas AE2 content remained unchanged. Enhanced activity and expression of Mrp2 was confirmed by analyzing the excretion rate of dinitrophenyl S-glutathione, an exogenous substrate of Mrp2, in isolated hepatocytes and by immunofluorescence microscopy, respectively. We conclude that SL increased bile flow mainly by increasing the biliary secretion of glutathione species and HCO(3)(-); increased expression of Mrp2 is also involved.

Estradiol-17β-d-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 μmol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 μmol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3–4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process.

Adriamycin (ADR) is a chemotherapeutic agent useful in treating various cancers. ADR is a quinone-containing anthracycline chemotherapeutic and is known to produce reactive oxygen species (ROS) in heart. Application of this drug can have serious side effects in various tissues, including brain, apart from the known cardiotoxic side effects, which limit the successful use of this drug in treatment of cancer. Neurons treated with ADR demonstrate significant protein oxidation and lipid peroxidation. Patients under treatment with this drug often complain of forgetfulness, lack of concentration, dizziness (collectively called somnolence or sometimes called chemobrain). In this study, we tested the hypothesis that ADR induces oxidative stress in brain. Accordingly, we examined the in vivo levels of brain protein oxidation and lipid peroxidation induced by i.p. injection of ADR. We also measured levels of the multidrug resistance-associated protein (MRP1) in brain isolated from ADR- or saline-injected mice. MRP1 mediates ATP-dependent export of cytotoxic organic anions, glutathione S-conjugates and sulphates. The current results demonstrated a significant increase in levels of protein oxidation and lipid peroxidation and increased expression of MRP1 in brain isolated from mice, 72 h post i.p injection of ADR. These results are discussed with reference to potential use of this redox cycling chemotheraputic agent in the treatement of cancer and its chemobrain side effect in brain.

Mari Golub, Lucio Costa, Kevin Crofton, Deborah Frank, Peter Fried, Beth Gladen, Rogene Henderson, Erica Liebelt, Shari Lusskin, Sue Marty, Andrew Rowland, John Scialli and Mary Vore California Environment Protection Agency, Sacramento, California University of Washington, Seattle, Washington U.S. Environmental Protection Agency, Research Triangle Park, North Carolina Boston Medical Center, Boston, Massachusetts Carleton University, Ottawa, Ontario National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina Lovelace Respiratory Research Institute, Albuquerque, New Mexico University of Alabama at Birmingham School of Medicine, Birmingham, Alabama New York University School of Medicine, New York, New York The Dow Chemical Company, Midland, Michigan University of New Mexico, Albuquerque, New Mexico Phoenix, Arizona University of Kentucky, Lexington, Kentucky

beta-estradiol 17-(beta-D-glucuronide) (E217G) is a well known cholestatic agent and substrate of multidrug resistance-associated protein 2 (Mrp2), whereas beta-estradiol 3-(beta-D-glucuronide) (E23G) is a noncholestatic regioisomer of E217G with unknown transport properties. The purpose of this study was to compare and contrast the Mrp2-mediated transport of E217G and E23G. The full coding region of rat Mrp2 was cloned into the baculovirus genome, the recombinant baculovirus used to infect Sf9 cells, and ATP-dependent transport of 3H-E23G and 3H-E217G in Sf9 cell membranes was characterized. Mrp2 transported E23G into an osmotically sensitive space, requiring ATP, with S50=55.7 microM, Vmax=326 pmol.mg(-1).min(-1), and a Hill coefficient of 0.88. ATP-dependent Mrp2-mediated E217G transport was markedly stimulated at high E217G concentrations, consistent with positive cooperativity (Hill coefficient 1.5). E217G (5-125 microM) increased S50 but not Vmax for E23G transport, consistent with competitive inhibition. E23G (0.4-400 microM) completely, potently (IC50=14.2 microM), and competitively inhibited E217G transport, but E217G (0.01-250 microM) inhibited only 53% of E23G transport (IC50=33.4 microM). Estriol 16alpha-(beta-D-glucuronide) potently and completely inhibited transport of E23G (IC50=2.23 microM), as did beta-estradiol 3-sulfate 17-(beta-D-glucuronide) (5-50 microM). In summary, E217G binds not only to an Mrp2 transport site, but also to an allosteric site that activates Mrp2 with positive cooperativity, thus activating its own transport and potentially that of other Mrp2 substrates, such as E23G. The noncholestatic E23G is an Mrp2 substrate and competes with E217G for transport, but does not activate the allosteric site.

Estradiol-17beta-D-glucuronide (E2-17G) induces an acute but reversible inhibition of bile flow after its intravenous administration to rats, due in part to the endocytic retrieval of the canalicular multidrug resistance-associated transporter protein 2 and the bile salt export pump, transporters that contribute to bile flow. Decreased bile salt-independent bile flow (BSIF) is also involved and persists during the phase of recovery from cholestasis. Because glutathione and HCO3- are major contributors to BSIF, we evaluated changes in their biliary excretion and the hepatic content of total glutathione during E2-17G-induced cholestasis. E2-17G acutely decreased bile flow and biliary excretion of total glutathione by about 80%; glutathione excretion was still inhibited at 80 min and 120 min, even though bile flow was partially and totally restored, respectively. Neither liver glutathione content nor the proportions of oxidized glutathione in bile and liver were affected by E2-17G at any time. HCO3- concentrations in bile were unchanged, so that secretion paralleled variations in bile flow. In the isolated perfused liver, addition of E2-17G decreased both bile flow and the biliary concentration of glutathione, whereas addition of its noncholestatic isomer estradiol-3-D-glucuronide (E2-3G) did not inhibit bile flow, but significantly reduced the concentration of glutathione in bile. The bile:liver concentration ratios of glutathione were significantly decreased in vivo by E2-17G and in the perfused liver by E2-17G and E2-3G. These data indicate that E2-17G cis-inhibits the canalicular transport of glutathione and thus contributes to the cholestatic effect by inhibiting BSIF.

The expression of hepatic multidrug resistance-associated protein (Mrp)1, 2, 3, and 6 and organic anion transporting polypeptides (Oatp)1 and 2 were examined in control and 20- to 21-day pregnant rats. Western analysis showed that expression of Oatp2 was decreased 50% in pregnancy, whereas expression of Oatp1 did not change. Expression of Mrp2 protein determined by Western analysis of total liver homogenate decreased to 50% of control levels in pregnant rats, consistent with studies using plasma membranes. Confocal immunohistochemistry showed that Mrp2 expression was confined to the canalicular membrane in both control and pregnant rats and was not detectable in intracellular compartments. In isolated perfused liver, the biliary excretion of 2,4-dintrophenyl-glutathione was significantly decreased in pregnancy, consistent with decreased expression of Mrp2. The expression of the basolateral transporter Mrp1 was not altered in pregnancy, whereas expression of Mrp6 mRNA was decreased by 60%. Expression of Mrp3 was also decreased by 50% in pregnant rat liver, indicating differential regulation of Mrp isoforms in pregnancy. These data also demonstrate that decreased Mrp2 expression is not necessarily accompanied by increased Mrp3 expression.

Estradiol-17β- D -glucuronide (E 2 17G), an endogenous metabolite of estradiol, induces a potent dose-dependent and reversible inhibition of bile flow in the rat. We analyzed the effect of a single dose of E 2 17G (15 μmol/kg, intravenously) to female rats on bile flow and the endocytic retrieval and function of the canalicular multidrug resistance-associated protein 2 (Mrp2) and the effect of pretreatment with dibutyryl-cyclic AMP (DBcAMP; 20 μmol/kg) on these measures. Bile flow was maximally inhibited by 85% within 10 minutes of E 2 17G and returned to 50% and 100% of control levels within 75 and 120 minutes, respectively. Western analysis of total homogenates and mixed plasma and intracellular membranes suggested partial internalization of Mrp2 during the acute phase of cholestasis at 20 minutes and during the period of recovery from cholestasis at 75 minutes, which returned to control levels by 180 minutes after E 2 17G. Confocal analysis confirmed Western studies and demonstrated endocytic retrieval of Mrp2 from the canalicular membrane into pericanalicular and intracellular domains. The biliary concentration and excretion of the model Mrp2 substrate, dinitrophenyl-S-glutathione (DNP-SG), was impaired in parallel with the extent of Mrp2 retrieval. Pretreatment with DBcAMP partially protected against maximal E 2 17G cholestasis and the endocytic retrieval and decreased function of Mrp2 at 20 minutes and significantly accelerated the exocytic insertion of Mrp2 into the canalicular membrane and the recovery of bile flow and biliary excretion of DNP-SG. In conclusion, these data indicate that E 2 17G induces endocytic internalization of Mrp2, which occurs in parallel with decreased bile flow and Mrp2 transport activity. (H EPATOLOGY 2002;35:1409-1419.)

The expression and activity of the apical ileal sodium-dependent bile acid transporter (asbt) was examined in the small intestine of control, pregnant, and lactating postpartum rats 2, 12, and 21 days after delivery. Western blot analysis of brush border membrane vesicles (BBMV) prepared from different regions of the small intestine demonstrated that expression of asbt was maximal in the most distal segments for all experimental groups, was not substantially affected in pregnant and 2-day postpartum rats, and was significantly increased in 12- and 21-day postpartum rats. Analysis of mRNA suggested that asbt protein was regulated at the posttranscriptional level in postpartum rats. Increased expression of asbt protein postpartum was maximal (∼2-fold) in the proximal region of the ileum, consistent with a 60% increase in taurocholate (TC) transport in BBMV from the proximal ileum in 14- to 21-day postpartum rats relative to control rats. Absorption of TC, determined from the intact proximal ileum using an intestinal loop model, demonstrated a 30% increase in TC uptake per unit weight of tissue in 14- to 21-day postpartum rats relative to control rats. Together with the marked increase in intestinal mass observed at peak lactation, these data indicate a significant increase in asbt-mediated reclamation of bile acids in the intestine of lactating rats.

We analyzed the expression of multidrug resistance-associated protein 2 (mrp2) in the small intestine of control female rats and in rats during late pregnancy (19–20 days of pregnancy) and lactation (2–4, 10–14, and 21 days after delivery). Western blot analysis was performed on brush-border membranes prepared from different regions of the small intestine. Expression of mrp2 was maximal in the proximal segments for all experimental groups, was preserved in pregnant rats, and increased by 100% in postpartum rats by late lactation with respect to control animals. Northern blot analysis of mrp2 mRNA revealed a positive correlation with protein levels. Transport of S-glutathione-dinitrophenol (DNP-SG) from the intestinal cell to the lumen was analyzed in the everted intestinal sac model. Secretion of DNP-SG was not altered in pregnant rats but increased in lactating animals by late lactation. Intestinal mrp2 mRNA, protein, and transport activity are increased in lactating rats, suggesting that this may represent an adaptive mechanism to minimize the toxicity of dietary xenobiotics in response to increased postpartum food consumption.

The promoting effects of polychlorinated biphenyls (PCBs) have been studied extensively in a variety of two-stage carcinogenesis models. However, the molecular mechanisms responsible for the promotion effects of PCBs have not been elucidated. We measured the effect of PCBs on DNA-binding proteins involved in cell proliferation and transformation. Male Sprague-Dawley rats were injected intraperitoneally with mono-, di-, tri-, tetra-, or hexachlorobiphenyls (300 mmol /kg/d) each day for 4 d and killed 4 h after the last injection. To detect alterations in nuclear proteins that could explain the tumor-promoter activity of PCBs, liver nuclear extracts were analyzed by electrophoretic mobility shift assays. Electrophoretic mobility shift assay analysis of signal transducers and activators of transcription (STAT)‐binding activity to a consensus g-interferon‐activated sequence (GAS) element was compared in liver nuclear extracts from treated rats. STAT-binding activity was eightfold to tenfold higher in nuclear extracts from animals treated with 2,4,4 0 -trichloro- (PCB 28) and 2,2 0 ,4,4 0 ,5,5 0 -hexachlorobiphenyl (PCB 153). Analysis of the protein complex binding to the GAS element, with antibodies specific for STAT3, STAT5, and STAT6, indicated that the protein complex was made up of STAT5 and STAT6 proteins. HepG2 cells transiently transfected with a luciferase reporter gene construct containing many STAT5 binding sites were treated with PCB 28 and PCB 153. PCB 28 stimulated a greater than 25-fold increase in luciferase activity at the highest concentration tested, 1.0 mg/mL. However, enhanced luciferase activity did not occur with PCB 153 treatment. 4-Chlorobiphenyl (PCB 3), PCB 28, and PCB 153 treatment of Sprague-Dawley rats resulted in a large increase in protein binding to a consensus activated protein-1 (AP-1) element. However, 3,4-dichlorobiphenyl (PCB 12) and 3,3 0 ,4,4 0 -tetrachlorobiphenyl (PCB 77) treatments did not increase AP-1 transcription activity. Further analysis of the proteins binding to the AP-1 consensus sequence with antibodies specific for c-fos, junD, and junB indicated that the protein composition consists of junD proteins. These data showed functional differences between noncoplanar and coplanar PCBs with respect to STAT activation and AP-1‐DNA binding. fl 2001 Wiley-Liss, Inc.