Induction of P-glycoprotein expression and activity by prolactin in female rat liver.

AIM: P-glycoprotein (P-gp) plays a critical role in the excretion of xenobiotics into bile. Previous studies have demonstrated that prolactin (PRL) regulates biotransformation and bile salt transport. Here we investigate whether the capability of the liver to transport xenobiotics into bile is altered in hyperprolactinemic states studying the modulation of hepatic P-gp by PRL. METHODS: We used lactating post-partum rats (PP), as a model of physiological hyperprolactinemia (15 and 21 days after delivery: PP15 and PP21, respectively), and ovariectomized rats treated with PRL (300 µg/day, 7 days, via osmotic minipumps, OVX + PRL). Hepatic P-gp expression and activity were evaluated by western blotting and using rhodamine 123 as substrate in vivo, respectively. Since P-gp is encoded by Mdr1a and Mdr1b in rodents, we quantified their expression by qPCR in primary hepatocyte cultures exposed to 0.1 µg/ml of PRL after 12 h. To further study the mechanism of hepatic P-gp modulation by PRL, hepatocytes were pretreated with actinomycin D and then exposed to PRL (0.1 µg/ml) for 12 h. KEY FINDINGS: We found increased hepatic P-gp protein expression and activity in PP15 and OVX + PRL. Also, a significant increase in Mdr1a and Mdr1b mRNA levels was observed in primary hepatocyte cultures exposed to PRL, pointing out the hormone direct action. Actinomycin D prevented these increases, confirming a transcriptional up-regulation of P-gp by PRL. SIGNIFICANCE: These findings suggest the possibility of an increased biliary excretion of xenobiotics substrates of P-gp, including therapeutic agents, affecting their pharmaco/toxicokinetics in hyperprolactinemic situations.

Protective effect of genistein pre-treatment on paraquat hepatotoxicity in rats.

Paraquat (PQ), an herbicide widely used in agriculture, is considered a highly toxic compound. In hepatocytes, P-glycoprotein (P-gp/Abcb1) is a canalicular transporter involved in PQ extrusion from the cell. Previously, we demonstrated that genistein (GNT) induces P-gp in rat liver. In this study, the protective role of GNT pretreatment towards hepatic damage in a model of acute intoxication with PQ in rats, was investigated. Wistar rats were randomized in 4 groups: Control, GNT (5 mg/kg/day sc, 4 days), PQ (50 mg/kg/day ip, last day) and GNT+ PQ. Hepatic lipoperoxidation (LPO) was evaluated by the thiobarbituric acid reactive substances method. Hepatic levels of 4-hydroxynonenal protein adducts (4-HNEp-add) and glutathione-S-transferase alpha (GSTα) protein expression were evaluated by Western blotting. Hepatic glutathione levels and plasma levels of alanine transaminase (ALT) and aspartate transaminase (AST) were also measured. Biliary excretion of PQ was studied in vivo and in isolated perfused liver. PQ was quantified by HPLC. PQ significantly increased AST and ALT activities, malondialdehyde and 4-HNEp-add levels, whereby pretreatment with GNT ameliorated this effect. PQ biliary excretion remained unchanged after treatments in both experimental models. Hepatic GSTα expression was augmented in GNT group. GNT pretreatment increased hepatic glutathione levels in PQ + GNT group. These results agree with the lower content of 4-HNEp-adds in GNT + PQ group respect to PQ group. Unexpectedly, increased activity of P-gp did not enhance PQ biliary excretion. Thus, GNT protective mechanism is likely through the induction of GSTα which results in increased 4-HNE metabolism before formation of protein adducts.

Up-regulation of ATP-binding cassette transporters in the THP-1 human macrophage cell line by the antichagasic benznidazole.

The effect of benznidazole (BZL) on the expression and activity of P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 2 (MRP2, ABCC2), the two major transporters of endogenous and exogenous compounds, was evaluated in differentiated THP-1 cells. BZL induced P-gp and MRP2 proteins in a concentration-dependent manner. The increase in mRNA levels of both transporters suggests transcriptional regulation. P-gp and MRP2 activities correlated with increased protein levels. BZL intracellular accumulation was significantly lower in BZL-pre-treated cells than in control cells. PSC833 (a P-gp inhibitor) increased the intracellular BZL concentration in both pre-treated and control cells, confirming P-gp participation in BZL efflux.

Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance.

The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 µM) for 48 h exhibited a dose-response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics.

Regulation of multidrug resistance proteins by genistein in a hepatocarcinoma cell line: impact on sorafenib cytotoxicity.

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 µM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 µM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 µM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 µM) and MRP2 induction by GNT (only at 10 µM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements.

Regulation of expression and activity of multidrug resistance proteins MRP2 and MDR1 by estrogenic compounds in Caco-2 cells. Role in prevention of xenobiotic-induced cytotoxicity.

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, 0.05pM to 5nM), or concentrations of genistein (GNT) associated with soy ingestion (0.1-10µM), 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 5pM EE and 1µM GNT for 48h 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 ERß, 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.

Modulation of biotransformation systems and ABC transporters by benznidazole in rats.

The effect of antichagasic benznidazole (BZL; 100 mg/kg body weight/day, 3 consecutive days, intraperitoneally) on biotransformation systems and ABC transporters was evaluated in rats. Expression of cytochrome P-450 (CYP3A), UDP-glucuronosyltransferase (UGT1A), glutathione S-transferases (alpha glutathione S-transferase [GST-α], GST-µ, and GST-π), multidrug-resistance-associated protein 2 (Mrp2), and P glycoprotein (P-gp) in liver, small intestine, and kidney was estimated by Western blotting. Increases in hepatic CYP3A (30%) and GST-µ (40%) and in intestinal GST-α (72% in jejunum and 136% in ileum) were detected. Significant increases in Mrp2 (300%) and P-gp (500%) proteins in liver from BZL-treated rats were observed without changes in kidney. P-gp and Mrp2 were also increased by BZL in jejunum (170% and 120%, respectively). In ileum, only P-gp was increased by BZL (50%). The activities of GST, P-gp, and Mrp2 correlated well with the upregulation of proteins in liver and jejunum. Plasma decay of a test dose of BZL (5 mg/kg body weight) administered intraduodenally was faster (295%) and the area under the concentration-time curve (AUC) was lower (41%) for BZL-pretreated rats than for controls. The biliary excretion of BZL was higher (60%) in the BZL group, and urinary excretion of BZL did not show differences between groups. The amount of absorbed BZL in intestinal sacs was lower (25%) in pretreated rats than in controls. In conclusion, induction of biotransformation enzymes and/or transporters by BZL could increase the clearance and/or decrease the intestinal absorption of coadministered drugs that are substrates of these systems, including BZL itself.

Acetaminophen inhibits intestinal p-glycoprotein transport activity.

Repeated acetaminophen (AP) administration modulates intestinal P-glycoprotein (P-gp) expression. Whether AP can modulate P-gp activity in a short-term fashion is unknown. We investigated the acute effect of AP on rat intestinal P-gp activity in vivo and in vitro. In everted intestinal sacs, AP inhibited serosal-mucosal transport of rhodamine 123 (R123), a prototypical P-gp substrate. R123 efflux plotted against R123 concentration adjusted well to a sigmoidal curve. Vmax decreased 50% in the presence of AP, with no modification in EC50, or slope, ruling out the possibility of inhibition to be competitive. Inhibition by AP was absent at 0°C, consistent with interference of the active transport of R123 by AP. Additionally, AP showed no effect on normal localization of P-gp at the apical membrane of the enterocyte and neither affected paracellular permeability. Consistent with absence of a competitive inhibition, two further strategies strongly suggested that AP is not a P-gp substrate. First, serosal-mucosal transport of AP was not affected by the classical P-gp inhibitors verapamil or Psc 833. Second, AP accumulation was not different between P-gp knock-down and wild-type HepG2 cells. In vivo intestinal absorption of digoxin, another substrate of P-gp, was assessed in the presence or absence of AP (100 µM). Portal digoxin concentration was increased by 214%, in average, by AP, as compared with digoxin alone. In conclusion, AP inhibited P-gp activity, increasing intestinal absorption of digoxin, a prototypical substrate. These results suggest that therapeutic efficacy of P-gp substrates can be altered if coadministered with AP.

Hormonal modulation of hepatic cAMP prevents estradiol 17ß-D-glucuronide-induced cholestasis in perfused rat liver.

BACKGROUND: Estradiol-17ß-D-glucuronide (E17G) induces cholestasis in vivo, endocytic internalization of the canalicular transporters multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11) being a key pathomechanism. Cyclic AMP (cAMP) prevents cholestasis by targeting these transporters back to the canalicular membrane. In hepatocyte couplets, glucagon and salbutamol, both of which increase cAMP, prevented E17G action by stimulating the trafficking of these transporters by different mechanisms, namely: glucagon activates a protein kinase A-dependent pathway, whereas salbutamol activates an exchange-protein activated by cAMP (Epac)-mediated, microtubule-dependent pathway. METHODS: The present study evaluated whether glucagon and salbutamol prevent E17G-induced cholestasis in a more physiological model, i.e., the perfused rat liver (PRL). Additionally, the preventive effect of in vivo alanine administration, which induces pancreatic glucagon secretion, was evaluated. RESULTS: In PRLs, glucagon and salbutamol prevented E17G-induced decrease in both bile flow and the secretory activity of Abcc2 and Abcb11. Salbutamol prevention fully depended on microtubule integrity. On the other hand, glucagon prevention was microtubule-independent only at early time periods after E17G administration, but it was ultimately affected by the microtubule disrupter colchicine. Cholestasis was associated with endocytic internalization of Abcb11 and Abcc2, the intracellular carriers being partially colocalized with the endosomal marker Rab11a. This effect was completely prevented by salbutamol, whereas some transporter-containing vesicles remained colocalized with Rab11a after glucagon treatment. In vivo, alanine administration increased hepatic cAMP and accelerated the recovery of bile flow and Abcb11/Abcc2 transport function after E17G administration. The initial recovery afforded by alanine was microtubule-independent, but microtubule integrity was required to sustain this protective effect. CONCLUSION: We conclude that modulation of cAMP levels either by direct administration of cAMP modulators or by physiological manipulations leadings to hormone-mediated increase of cAMP levels (alanine administration), prevents estrogen-induced cholestasis in models with preserved liver architecture, through mechanisms similar to those arisen from in vitro studies.

Sequential activation of classic PKC and estrogen receptor α is involved in estradiol 17ß-D-glucuronide-induced cholestasis.

UNLABELLED: Estradiol 17ß-D-glucuronide (E17G) induces acute cholestasis in rat with endocytic internalization of the canalicular transporters bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2). Classical protein kinase C (cPKC) and PI3K pathways play complementary roles in E17G cholestasis. Since non-conjugated estradiol is capable of activating these pathways via estrogen receptor alpha (ERα), we assessed the participation of this receptor in the cholestatic manifestations of estradiol glucuronidated-metabolite E17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHC). In both models, E17G activated ERα. In PRL, E17G maximally decreased bile flow, and the excretions of dinitrophenyl-glutathione, and taurocholate (Abcc2 and Abcb11 substrates, respectively) by 60% approximately; preadministration of ICI 182,780 (ICI, ERα inhibitor) almost totally prevented these decreases. In IRHC, E17G decreased the canalicular vacuolar accumulation of cholyl-glycylamido-fluorescein (Abcb11 substrate) with an IC50 of 91±1 µM. ICI increased the IC50 to 184±1 µM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Abcc2 substrate, glutathione-methylfluorescein. ICI also completely prevented E17G-induced delocalization of Abcb11 and Abcc2 from the canalicular membrane, both in PRL and IRHC. The role of ERα in canalicular transporter internalization induced by E17G was confirmed in ERα-knocked-down hepatocytes cultured in collagen sandwich. In IRHC, the protection of ICI was additive to that produced by PI3K inhibitor wortmannin but not with that produced by cPKC inhibitor Gö6976, suggesting that ERα shared the signaling pathway of cPKC but not that of PI3K. Further analysis of ERα and cPKC activations induced by E17G, demonstrated that ICI did not affect cPKC activation whereas Gö6976 prevented that of ERα, indicating that cPKC activation precedes that of ERα. CONCLUSION: ERα is involved in the biliary secretory failure induced by E17G and its activation follows that of cPKC.

Regulation of biotransformation systems and ABC transporters by benznidazole in HepG2 cells: involvement of pregnane X-receptor.

BACKGROUND: Benznidazole (BZL) is the only antichagasic drug available in most endemic countries. Its effect on the expression and activity of drug-metabolizing and transporter proteins has not been studied yet. METHODOLOGY/PRINCIPAL FINDINGS: Expression and activity of P-glycoprotein (P-gp), Multidrug resistance-associated protein 2 (MRP2), Cytochrome P450 3A4 (CYP3A4), and Glutathione S-transferase (GST) were evaluated in HepG2 cells after treatment with BZL. Expression was estimated by immunoblotting and real time PCR. P-gp and MRP2 activities were estimated using model substrates rhodamine 123 and dinitrophenyl-S-glutathione (DNP-SG), respectively. CYP3A4 and GST activities were evaluated through their abilities to convert proluciferin into luciferin and 1-chloro-2,4-dinitrobenzene into DNP-SG, respectively. BZL (200 µM) increased the expression (protein and mRNA) of P-gp, MRP2, CYP3A4, and GSTπ class. A concomitant enhancement of activity was observed for all these proteins, except for CYP3A4, which exhibited a decreased activity. To elucidate if pregnane X receptor (PXR) mediates BZL response, its expression was knocked down with a specific siRNA. In this condition, the effect of BZL on P-gp, MRP2, CYP3A4, and GSTπ protein up-regulation was completely abolished. Consistent with this, BZL was able to activate PXR, as detected by reporter gene assay. Additional studies, using transporter inhibitors and P-gp-knock down cells, demonstrated that P-gp is involved in BZL extrusion. Pre-treatment of HepG2 cells with BZL increased its own efflux, as a consequence of P-gp up-regulation. CONCLUSIONS/SIGNIFICANCE: Modifications in the activity of biotransformation and transport systems by BZL may alter the pharmacokinetics and efficiency of drugs that are substrates of these systems, including BZL itself.

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.

Induction of intestinal multidrug resistance-associated protein 2 (Mrp2) by spironolactone in rats.

The effect of spironolactone (SL) pretreatment (200micromol/kg b.w./day, 3 consecutive days) on intestinal multidrug resistance-associated protein 2 (Mrp2) was evaluated in rats. A significant increase in protein levels in upper regions of small intestine, where Mrp2 is mainly present, was detected by western blotting. Real time PCR studies suggest a transcriptional regulation. The administration of ketoconazole, a pregnane X receptor (PXR) antagonist, was able to prevent the increase in Mrp2 mRNA levels induced by SL. The serosal to mucosal transport of dinitrophenyl S-glutathione, a model substrate of Mrp2 was evaluated in jejunal sac model. The data indicate that SL increased Mrp2 activity, well correlating with its up-regulation. We conclude that SL is able to induce intestinal Mrp2 transcriptionally, PXR being a potential mediator. We propose that SL could be of potential therapeutic application particularly in situations of down-regulation of intestinal Mrp2.

Effect of repeated administration with subtoxic doses of acetaminophen to rats on enterohepatic recirculation of a subsequent toxic dose.

Development of resistance to toxic effects of acetaminophen (APAP) was reported in rodents and humans, though the mechanism is only partially understood. We examined in rats the effect of administration with subtoxic daily doses (0.2, 0.3, and 0.6g/kg, i.p.) of APAP on enterohepatic recirculation and liver toxicity of a subsequent i.p. toxic dose of 1g/kg, given 24h after APAP pre-treatment. APAP and its major metabolite APAP-glucuronide (APAP-Glu) were determined in bile, urine, serum and liver homogenate. APAP pre-treatment was not toxic, as determined by serum markers of liver damage and neither induced oxidative stress as demonstrated by assessment of ROS generation in liver or glutathione species in liver and bile. APAP pre-treatment induced a partial shift from biliary to urinary elimination of APAP-Glu after administration with the toxic dose, and decreased hepatic content and increased serum content of this conjugate, consistent with a marked up-regulation of its basolateral transporter Mrp3 relative to apical Mrp2. Preferential secretion of APAP-glu into blood decreased enterohepatic recirculation of APAP, thus attenuating liver exposition to the intact drug, as demonstrated 6h after administration with the toxic dose. The beneficial effect of interfering the enterohepatic recirculation was alternatively tested in animals receiving activated charcoal by gavage to adsorb APAP of biliary origin. The data indicated decreased liver APAP content and glutathione consumption. We conclude that selective up-regulation of Mrp3 expression by APAP pre-treatment may contribute to development of resistance to APAP hepatotoxicity, at least in part by decreasing its enterohepatic recirculation.

Regulation of expression and activity of rat intestinal multidrug resistance-associated protein 2 by cholestatic estrogens.

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.

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver.

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.

Hepatic synthesis and urinary elimination of acetaminophen glucuronide are exacerbated in bile duct-ligated rats.

Renal and intestinal disposition of acetaminophen glucuronide (APAP-GLU), a common substrate for multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), was assessed in bile duct-ligated rats (BDL) 7 days after surgery using an in vivo perfused jejunum model with simultaneous urine collection. Doses of 150 mg/kg b.w. (i.v.) or 1 g/kg b.w. (i.p.) of acetaminophen (APAP) were administered, and its glucuronide was determined in bile (only Shams), urine, and intestinal perfusate throughout a 150-min period. Intestinal excretion of APAP-GLU was unchanged or decreased (-58%) by BDL for the 150 mg and 1 g/kg b.w. doses of APAP, respectively. In contrast, renal excretion was increased by 200 and 320%, respectively. Western studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, whereas Mrp3 was substantially increased in liver and not affected in kidney or intestine. The global synthesis of APAP-GLU, determined as the sum of cumulative excretions, was higher in BDL rats (+51 and +110%) for these same doses of APAP as a consequence of a significant increase in functional liver mass, with no changes in specific glucuronidating activity. Expression of apical breast cancer resistance protein, which also transports nontoxic metabolites of APAP, was decreased by BDL in liver and renal cortex, suggesting a minor participation of this route. We demonstrate a more efficient hepatic synthesis and basolateral excretion of APAP-GLU followed by its urinary elimination in BDL group, the latter two processes consistent with up-regulation of liver Mrp3 and renal Mrp2.

Beneficial effect of spironolactone administration on ethynylestradiol-induced cholestasis in the rat: involvement of up-regulation of multidrug resistance-associated protein 2.

The effect of spironolactone (SL) administration on 17alpha-ethynylestradiol (EE)-induced cholestasis was studied, with emphasis on expression and activity of Mrps. Adult male Wistar rats were divided into the following groups: EE (5 mg/kg daily for 5 days, s.c.), SL (200 micromol/kg daily for 3 days, i.p.), EE+SL (same doses, SL administered the last 3 days of EE treatment), and controls. SL prevented the decrease in bile salt-independent fraction of bile flow induced by EE, in association with normalization of biliary excretion of glutathione. Western blot studies indicate that EE decreased the expression of multidrug resistance-associated protein 2 (Mrp2) by 41% and increased that of Mrp3 by 200%, whereas SL only affected Mrp2 expression (+60%) with respect to controls. The EE+SL group showed increased levels of Mrp2 and Mrp3 to the same extent as that registered for the individual treatments. Real-time polymerase chain reaction studies indicated that up-regulation of Mrp2 and Mrp3 by SL and EE, respectively, was at the transcriptional level. To estimate Mrp2 and Mrp3 activities, apical and basolateral excretion of acetaminophen glucuronide (APAP-glu), a common substrate for both transporters, was measured in the recirculating isolated perfused liver model. Biliary/perfusate excretion ratio was decreased in EE (-88%) and increased in SL (+36%) with respect to controls. Coadministration of rats with SL partially prevented (-53%) impairment induced by EE in this ratio. In conclusion, SL administration to EE-induced cholestatic rats counteracted the decrease in bile flow and biliary excretion of glutathione and APAP-glu, a model Mrp substrate, findings associated with up-regulation of Mrp2 expression.

Induction of rat intestinal P-glycoprotein by spironolactone and its effect on absorption of orally administered digoxin.

The effect of the diuretic spironolactone (SL) on expression and function of intestinal P-glycoprotein (P-gp), as well as its impact on intestinal absorption of digoxin, was explored. Rats were treated with daily doses of 200 micromol/kg b.wt. of SL intraperitoneally for 3 consecutive days. The small intestine was divided into four equal segments of approximately 25 cm, with segment I being the most proximal. Brush-border membranes were isolated and used in analysis of P-gp expression by Western blot analysis. P-gp content increased in the SL group by 526, 292, 210, and 622% over controls for segments I, II, III, and IV, respectively. Up-regulation of apical P-gp was confirmed by immunofluorescence microscopy. P-gp transport activity was explored in intestinal sacs prepared from segment IV using two different model substrates. Serosal to mucosal transport (efflux) of rhodamine 123 was 140% higher, and mucosal to serosal transport (absorption) of digoxin was 40% lower in the SL group, both indicating increased P-gp function. In vivo experiments showed that intestinal absorption of a single dose of digoxin administered p.o. was attenuated by SL pretreatment. Thus, concentration of digoxin in portal and peripheral blood was lower in SL versus control groups, as well as its accumulation in kidney and liver. Urinary excretion of digoxin was significantly decreased in the SL group, probably reflecting decreased systemic availability of digoxin for subsequent urinary elimination. We conclude that SL induces P-gp expression with potential impact on intestinal absorption of substrates with therapeutic application.

Hepatic and extrahepatic synthesis and disposition of dinitrophenyl-S-glutathione in bile duct-ligated rats.

The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for the multidrug resistance-associated proteins (Mrps), was assessed in bile duct-ligated (BDL) rats 1, 7, and 14 days after surgery, using an in vivo perfused jejunum model with simultaneous urine collection. A single i.v. dose of 30 micromol/kg b.wt. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its glutathione conjugate DNP-SG and dinitrophenyl cysteinyl glycine derivative, which is the result of gamma-glutamyl-transferase action on DNP-SG, were determined in urine and intestinal perfusate by high-performance liquid chromatography. Intestinal excretion of these metabolites was unchanged at day 1, and decreased at days 7 and 14 (-39% and -33%, respectively) after surgery with respect to shams. In contrast, renal excretion was increased by 114%, 150%, and 128% at days 1, 7, and 14. Western blot studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, these changes being maximal between days 7 and 14. Assessment of expression of basolateral Mrp3 at day 14 postsurgery indicated preserved levels in renal cortex, duodenum, jejunum, distal ileum, and colon. Analysis of expression of glutathione-S-transferases alpha, mu, and pi, as well as activity toward CDNB, indicates that formation of DNP-SG was impaired in liver, preserved in intestine, and increased in renal cortex. In conclusion, increased renal tubular conversion of CDNB to DNP-SG followed by subsequent Mrp2-mediated secretion into urine partially compensates for altered liver function in experimental obstructive cholestasis.