Hepatic Bile Formation: Canalicular Osmolarity and Paracellular and Transcellular Water Flow.

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 17ß-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.

Loss of multidrug resistance-associated protein 1 potentiates chronic doxorubicin-induced cardiac dysfunction in mice.

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.

Elevated glutathione is not sufficient to protect against doxorubicin-induced nuclear damage in heart in multidrug resistance-associated protein 1 (Mrp1/Abcc1) null mice.

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-naïve 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-naïve 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.

Abcb11 deficiency induces cholestasis coupled to impaired ß-fatty acid oxidation in mice.

The bile salt export pump (BSEP) is an ATP-binding cassette transporter that serves as the primary system for removing bile salts from the liver. In humans, deficiency of BSEP, which is encoded by the ABCB11 gene, causes severe progressive cholestatic liver disease from early infancy. In previous studies of Abcb11 deficiency in mice generated on a mixed genetic background, the animals did not recapitulate the human disease. We reasoned that ABCB11 deficiency may cause unique changes in hepatic metabolism that are predictive of liver injury. To test this possibility, we first determined that Abcb11 knock-out (KO) C57BL/6J mice recapitulate human deficiency. Before the onset of cholestasis, Abcb11 KO mice have altered hepatic lipid metabolism coupled with reduced expression of genes important in mitochondrial fatty acid oxidation. This was associated with increased serum free-fatty acids, reduced total white adipose, and marked impairment of long-chain fatty acid ß-oxidation. Importantly, metabolomic analysis confirmed that Abcb11 KO mice have impaired mitochondrial fatty acid ß-oxidation with the elevated fatty acid metabolites phenylpropionylglycine and phenylacetylglycine. These metabolic changes precede cholestasis but may be of relevance to cholestatic disease progression because altered fatty acid metabolism can enhance reactive oxygen species that might exacerbate cholestatic liver damage.

Induction of hepatic multidrug resistance-associated protein 3 by ethynylestradiol is independent of cholestasis and mediated by estrogen receptor.

Multidrug resistance-associated protein 3 (Mrp3; Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas multidrug resistance-associated protein 2 (Mrp2) is down-regulated. This study was undertaken to determine whether Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, noncholestatic dose of EE (5 mg/kg s.c.), and basal bile flow and the biliary excretion rate of bile salts and glutathione were measured 5 hours later. This treatment increased Mrp3 mRNA by 4-fold, detected by real-time polymerase chain reaction, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1-10 µM) for 5 hours exhibited a 3-fold increase in Mrp3 mRNA (10 µM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were incubated with an ER antagonist [7α,17ß-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI182/780), 1 µM], in addition to EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 minutes and expression of c-Jun, a well-known ER target gene, at 60 minutes, as detected by Western blotting of nuclear extracts. These increases were prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation and required participation of an ER, most likely ER-α, through its phosphorylation.

The G671V variant of MRP1/ABCC1 links doxorubicin-induced acute cardiac toxicity to disposition of the glutathione conjugate of 4-hydroxy-2-trans-nonenal.

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.

Effect of glucagon-like peptide 2 on hepatic, renal, and intestinal disposition of 1-chloro-2,4-dinitrobenzene.

The ability of the liver, small intestine, and kidney to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), was assessed in rats treated with glucagon-like peptide 2 (GLP-2, 12 µg/100 g b.wt. s.c. every 12 h for 5 consecutive days). An in vivo perfused jejunum model with simultaneous bile and urine collection was used. A single intravenous dose of 30 µmol/kg b.wt. 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its conjugate, DNP-SG, and dinitrophenyl cysteinyl glycine (DNP-CG), resulting from the action of γ-glutamyltransferase on DNP-SG, were determined in bile, intestinal perfusate, and urine by high-performance liquid chromatography. Tissue content of DNP-SG was also assessed in liver, intestine, and kidneys. Biliary excretion of DNP-SG+DNP-CG was decreased in GLP-2 rats with respect to controls. In contrast, their intestinal excretion was substantially increased, whereas urinary elimination was not affected. Western blot and real-time polymerase chain reaction studies revealed preserved levels of Mrp2 protein and mRNA in liver and renal cortex and a significant increase in intestine in response to GLP-2 treatment. Tissue content of DNP-SG detected 5 min after CDNB administration was decreased in liver, increased in intestine, and unchanged in kidney in GLP-2 versus control group, consistent with GLP-2-induced down-regulation of expression of glutathione transferase (GST) Mu in liver and up-regulation of GST-Alpha in intestine at both protein and mRNA levels. In conclusion, GLP-2 induced selective changes in hepatic and intestinal disposition of a common GST and Mrp2 substrate administered systemically that could be of pharmacological or toxicological relevance under therapeutic treatment conditions.

Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes.

BACKGROUND: Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. RESULTS: A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p < 0.05 for the overall physiologic state effect (lactation vs. control), and a within tissue pairwise comparison of p < 0.01. The proportion of false positives, an estimate of the ratio of false positives in the list of differentially expressed genes, was calculated for each tissue. The number of differentially expressed genes was 420 in the liver, 337 in the duodenum, 402 in the jejunum, and 523 in the ileum. The list of differentially expressed genes was in turn analyzed by Ingenuity Pathways Analysis (IPA) to detect biological pathways that were overrepresented. In all tissues, sterol regulatory element binding protein (Srebp)-regulated genes involved in cholesterol synthesis showed increased mRNA expression, with the fewest changes detected in the jejunum. We detected increased Scap mRNA in the liver only, suggesting an explanation for the difference in response to lactation between the liver and small intestine. Expression of Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC) and Adenosine Triphosphate Binding Cassette (ABC) superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175. CONCLUSIONS: We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and jejunum) was also noted, as well as differential expression of transporters that likely play a key role in nutrient uptake.

Functional analysis of nonsynonymous single nucleotide polymorphisms of multidrug resistance-associated protein 2 (ABCC2).

BACKGROUND: 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. OBJECTIVES: To characterize the transport function of human wild-type (WT) MRP2 and four SNP variants, S789F, A1450T, V417I, and T1477M. METHODS: 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-17ß-glucuronide (E(2)17G), and tauroursodeoxycholic acid (TUDC) were determined in Sf9-derived plasma membrane vesicles. Transport activity was normalized for expression level. RESULTS: 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. CONCLUSION: 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.

Mechanisms for increased expression of cholesterol 7alpha-hydroxylase (Cyp7a1) in lactating rats.

UNLABELLED: Cholesterol 7alpha-hydroxylase (Cyp7a1) and the bile acid pool size are increased 2 to 3-fold in lactating postpartum rats. We investigated the interaction of nuclear receptors with the Cyp7a1 proximal promoter and the expression of regulatory signaling pathways in postpartum rats at day 10 (PPd10) versus female controls to identify the mechanisms of increased expression of Cyp7a1, which is maximal at 16 hours. Liver X receptor (LXRalpha) and RNA polymerase II (RNA Pol II) recruitment to Cyp7a1 chromatin were increased 1.5- and 2.5-fold, respectively, at 16 hours on PPd10. Expression of nuclear receptors farnesoid X receptor (FXR), LXRalpha, liver receptor homolog (LRH-1), hepatocyte nuclear factor 4alpha (HNF4alpha), and short heterodimer partner (SHP) messenger RNA (mRNA) and coactivator peroxisome proliferators-activated receptor gamma coactivator-1alpha (PGC-1alpha) mRNA was unchanged in PPd10 versus controls at 16 hours, whereas chicken ovalbumin upstream transcription factor II (COUP-TFII) was decreased 40% at 16 hours. Investigation of a repressive signaling pathway, the c-Jun-N-terminal kinase (JNK) signaling pathway in PPd10 versus controls, showed decreased mRNA expression of hepatocyte growth factor (HGF; decreased 60% at 16 hours) and tyrosine kinase receptor c-Met (decreased 44%-50% at 16 hours), but these were not accompanied by decreased expression of phosphorylated c-Jun. Importantly, expression of fibroblast growth factor 15 (FGF15) mRNA in the ileum was decreased 70% in PPd10 versus controls, whereas phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Erk1/2) protein expression in liver was decreased 88% at 16 hours. CONCLUSION: The increased recruitment of LXRalpha, a Cyp7a1 stimulatory pathway, and decreased expression of FGF15 and phosphorylated Erk1/2, a Cyp7a1 repressive pathway, combined to increase Cyp7a1 expression during lactation.

Phosphoinositide 3-kinase/protein kinase B signaling pathway is involved in estradiol 17ß-D-glucuronide-induced cholestasis: complementarity with classical protein kinase C.

UNLABELLED: Estradiol 17ß-D-glucuronide (E(2)17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E(2)17G-induced cholestasis. E(2)17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E(2)17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E(2)17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E(2)17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E(2)17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [(3)H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. CONCLUSION: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E(2)17G through sustained internalization of canalicular transporters endocytosed via cPKC.

The 5'-untranslated region of multidrug resistance associated protein 2 (MRP2; ABCC2) regulates downstream open reading frame expression through translational regulation.

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.

ABCG5/ABCG8-independent biliary cholesterol excretion in lactating rats.

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.

Induction of intestinal multidrug resistance-associated protein 2 by glucagon-like Peptide 2 in the rat.

The effects of glucagon-like peptide 2 (GLP-2) on expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2; Abcc2) and glutathione transferase (GST) were evaluated. After GLP-2 treatment (12 µg/100 g b.wt. s.c., every 12 h, for 5 consecutive days), Mrp2 and the α class of GST proteins and their corresponding mRNAs were increased, suggesting a transcriptional regulation. Mrp2 was localized at the apical membrane of the enterocyte in control and GLP-2 groups, as detected by confocal immunofluorescence microscopy. As a functional assay, everted intestinal sacs were incubated in the presence of 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl-S-glutathione (DNP-SG; model Mrp2 substrate), was detected in the same compartment by high-performance liquid chromatography. A significant increase in apical secretion of DNP-SG was detected in the GLP-2 group, consistent with simultaneous up-regulation of Mrp2 and GST. GLP-2 also promoted an increase in cAMP levels as detected in homogenates of intestinal mucosa. Treatment of rats with 2',3'-dideoxyadenosine (DDA), a specific inhibitor of adenylyl cyclase, abolished the increase in cAMP levels and Mrp2 protein promoted by GLP-2, suggesting cAMP as a mediator of Mrp2 modulation. Increased expression of Mrp2 and cAMP levels in response to GLP-2 occurred not only at the tip but also at the middle region of the villus, where constitutive expression of Mrp2 is normally low. In conclusion, our study suggests a role for GLP-2 in the prevention of cell toxicity of the intestinal mucosa by increasing Mrp2 chemical barrier function.

Hepatobiliary disposition of 3alpha,6alpha,7alpha,12alpha-tetrahydroxy-cholanoyl taurine: a substrate for multiple canalicular transporters.

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 (K(m) = 0.13 mM) than for mMdr1a (K(m) = 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.

Chemo brain (chemo fog) as a potential side effect of doxorubicin administration: role of cytokine-induced, oxidative/nitrosative stress in cognitive dysfunction.

Doxorubicin (ADRIAMYCIN, RUBEX) is a chemotherapeutic agent that is commonly administered to breast cancer patients in standard chemotherapy regimens. As true of all such therapeutic cytotoxic agents, it can damage normal, noncancerous cells and might affect biochemical processes in a manner that might lead to, or contribute to, chemotherapy-induced cognitive deficits when administered either alone or in combination with other agents.

Mrp1 localization and function in cardiac mitochondria after doxorubicin.

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 muM) 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.