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.

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.

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.

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 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.

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.

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.

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.

Mechanisms involved in spironolactone-induced choleresis in the rat. Role of multidrug resistance-associated protein 2.

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.

Involvement of Mrp2 in hepatic and intestinal disposition of dinitrophenyl-S-glutathione in partially hepatectomized rats.

The ability of the liver and small intestine for secretion of dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), into bile and lumen, respectively, as well as expression of Mrp2 in both tissues, were assessed in 70-75% hepatectomized rats. An in vivo perfused intestinal model was used. A single i.v. dose of 30 micromol/kg b.w. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered and its glutathione conjugate, DNP-SG, was determined by HPLC in bile and intestinal perfusate. One and seven days after hepatectomy, biliary excretion of DNP-SG was decreased by 90 and 50% with respect to shams, respectively, when expressed per mass unit. In contrast, intestinal excretion was increased by 63% or unchanged one and seven days post-hepatectomy, respectively. Tissue content of DNP-SG 5 min after CDNB administration was substantially decreased in liver and significantly increased in intestine, one day post-hepatectomy. Western and immunofluorescence studies revealed preserved levels and localization of Mrp2 in both tissues from hepatectomized animals, irrespective of the time analyzed. In spite of preserved expression of Mrp2, the higher availability of DNP-SG in intestinal cells, likely as a consequence of increased glutathione-S-transferase-mediated conjugation of CDNB, may explain the in vivo findings. Further experiments in isolated hepatocytes suggested that decreased synthesis of DNP-SG rather than altered canalicular transport is responsible for the substantial impairment in excretion of this compound into bile. Taken together, these results indicate that the intestine may partially compensate for liver DNP-SG disposition, particularly shortly after surgery, while liver capability is recovering.

Effect of spironolactone on the expression of rat hepatic UDP-glucuronosyltransferase.

Spironolactone (SL) increases the glucuronidation rate of several compounds. We analyzed the molecular basis of changes occurring in major rat liver UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform of family 2, in response to SL. UGT activity toward bilirubin, ethynylestradiol and p-nitrophenol was assayed in native and activated microsomes. Protein and mRNA levels were determined by Western and Northern blotting. The lipid composition and physicochemical properties of the microsomal membrane were also analyzed. Glucuronidation rates of bilirubin and ethynylestradiol (at both 3-OH and 17 beta-OH positions), determined in UDP-N-acetylglucosamine-activated membranes, were increased in SL group. Western blot analysis revealed increased levels of UGT1A1 and 1A5 (bilirubin and 3-OH ethynylestradiol conjugation), and 2B1 (17 beta-OH ethynylestradiol conjugation). Northern blot studies suggested transcriptional regulation by the steroid. Analysis of UGT activity in native vs. alamethicin-activated microsomes indicated increased latency, which was not associated to changes in physicochemical properties of the microsomal membrane. p-Nitrophenol glucuronidation rate and mRNA and protein levels of UGT1A6, the main isoform conjugating planar phenols, were not affected by the inducer. The data suggest transcriptional regulation of specific isoforms of hepatic UGT by SL, thus explaining previously reported increases in UGT activity toward selective substrates.

Co-regulation of expression of phase II metabolizing enzymes and multidrug resistance-associated protein 2.

Treatment of experimental animals with prototypical enzyme inducers represents a useful tool to characterize the role of different isozymes in drug metabolism and to improve our knowledge on factors regulating their synthesis at the transcriptional level. The effect of model enzyme inducers on phase II (conjugating) enzyme families, including UDP-glucuronosyltransferase's and glutathione-S-transferase's, has been well characterized in rodent liver. More recently, the effect of inducers on the expression of canalicular multidrug resistance-associated protein 2 (Mrp2) has been focused upon. The identification of a number of conjugated drugs as Mrp2 substrates suggests that both the conjugation and transport systems act coordinately to improve drug elimination from the body. We provide evidence about circumstances resulting in the simultaneous upregulation of phase II enzymes and Mrp2 in hepatic and extrahepatic tissues, most likely involving activation of common nuclear receptors (e.g. FXR, PXR). Additionally, we provide an analysis of examples of drug-induced toxicity leading to the simultaneous downregulation of both systems. Potential therapeutic strategies based on the modulation of expression of these systems are also briefly commented upon.

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.

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.

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.

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.

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.

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.

Ethynylestradiol increases expression and activity of rat liver MRP3.

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.

Galactosamine prevents ethinylestradiol-induced cholestasis.

Ethinylestradiol (EE) induces intrahepatic cholestasis in experimental animals being its derivative, ethinylestradiol 17beta-glucuronide, a presumed mediator of this effect. To test whether glucuronidation is a relevant step in the pathogenesis of cholestasis induced by EE (5 mg/kg b.wt. s.c. for 5 consecutive days), the effect of simultaneous administration of galactosamine (200 mg/kg b.wt. i.p.) on biliary secretory function was studied. A single injection of this same dose of galactosamine was able to decrease hepatic UDP-glucuronic acid (UDP-GA) levels by 85% and excretion of EE-17beta-glucuronide after administration of a tracer dose of [3H]EE by 40%. Uridine (0.9 g/kg b.wt. i.p.) coadministration reverted the effect of galactosamine on hepatic UDP-GA levels and restored the excretion of [3H]EE-17beta-glucuronide. When administered for 5 days, galactosamine itself did not alter any of the serum markers of liver injury studied (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) or biliary secretory function. When coadministered with EE, galactosamine partially prevented the impairment induced by this estrogen in total bile flow, the bile-salt-independent fraction of bile flow, basal bile salt secretion, and the secretory rate maximum of tauroursodeoxycholate. Uridine coadministration partially prevented galactosamine from exerting its anticholestatic effects. In conclusion, galactosamine administration partially prevented EE-induced cholestasis by a mechanism involving decreased UDP-GA availability for subsequent formation of EE 17beta-glucuronide. The evidence thus supports the hypothesis that EE 17beta-glucuronide is involved in the pathogenesis of EE cholestasis.