RESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Fígado/efeitos dos fármacos , Fígado/metabolismo , Prolactina/metabolismo , Prolactina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Lactação/efeitos dos fármacos , Lactação/metabolismo , Ovariectomia , Ratos , Ratos Wistar , OvinosRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Nitroimidazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Subfamília B de Transportador de Cassetes de Ligação de ATP/efeitos dos fármacos , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Linhagem Celular , Doença de Chagas/metabolismo , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Nitroimidazóis/farmacologia , Tripanossomicidas/farmacologia , Regulação para CimaRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Etinilestradiol/farmacologia , Genisteína/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Proteínas de Neoplasias/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Células CACO-2 , Dinitroclorobenzeno/toxicidade , Relação Dose-Resposta a Droga , Antagonistas de Estrogênios/farmacologia , Receptor beta de Estrogênio/genética , Etinilestradiol/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Genisteína/administração & dosagem , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Paraquat/toxicidade , RNA Mensageiro/metabolismo , Glycine max/química , Regulação para Cima/efeitos dos fármacos , Xenobióticos/toxicidadeRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Acetaminofen/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Ciclosporinas/farmacologia , Digoxina/farmacologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Células Hep G2 , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Wistar , Rodamina 123/farmacologia , Verapamil/farmacologiaRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Nitroimidazóis/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Western Blotting , Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/metabolismo , Absorção Intestinal/efeitos dos fármacos , Isoenzimas/metabolismo , Masculino , Nitroimidazóis/sangue , Nitroimidazóis/farmacocinética , RatosRESUMO
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.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Albuterol/uso terapêutico , Colestase/prevenção & controle , AMP Cíclico/agonistas , Estradiol , Glucagon/uso terapêutico , Hormônios/uso terapêutico , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Alanina/uso terapêutico , Animais , Biomarcadores/metabolismo , Colestase/etiologia , Colestase/metabolismo , AMP Cíclico/metabolismo , Feminino , Fígado/metabolismo , Fígado/fisiopatologia , Ratos , Ratos Wistar , Resultado do Tratamento , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Colestase/induzido quimicamente , Colestase/metabolismo , Estradiol/análogos & derivados , Receptor alfa de Estrogênio/metabolismo , Proteína Quinase C/metabolismo , Animais , Carbazóis/farmacologia , Células Cultivadas , Estradiol/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Feminino , Fulvestranto , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Antiprotozoários/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Nitroimidazóis/metabolismo , Receptores de Esteroides/metabolismo , Biotransformação , Western Blotting , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Redes e Vias Metabólicas/genética , Receptor de Pregnano X , Reação em Cadeia da Polimerase em Tempo Real , Regulação para CimaRESUMO
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.
Assuntos
Dinitroclorobenzeno/farmacocinética , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Jejuno/metabolismo , Rim/metabolismo , Fígado/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bile/metabolismo , Dinitrobenzenos/metabolismo , Dinitroclorobenzeno/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Jejuno/efeitos dos fármacos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Wistar , Regulação para Cima/efeitos dos fármacos , gama-Glutamiltransferase/metabolismoRESUMO
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.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Espironolactona/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Polaridade Celular , Dinitroclorobenzeno/metabolismo , Dinitroclorobenzeno/farmacocinética , Glutationa/análogos & derivados , Glutationa/análise , Intestino Delgado/metabolismo , Masculino , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Esteroides/antagonistas & inibidoresRESUMO
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.
Assuntos
Colestase/metabolismo , Estrogênios/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Mucosa Intestinal/metabolismo , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ratos , Ratos WistarRESUMO
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.
Assuntos
Dapsona/farmacologia , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Feminino , Glucuronosiltransferase/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Acetaminofen/análogos & derivados , Acetaminofen/metabolismo , Fígado/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetaminofen/administração & dosagem , Acetaminofen/urina , Animais , Ductos Biliares/cirurgia , Relação Dose-Resposta a Droga , Mucosa Intestinal/metabolismo , Rim/metabolismo , Ligadura , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ratos , Ratos WistarRESUMO
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.
Assuntos
Colestase Intra-Hepática/prevenção & controle , Etinilestradiol/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Espironolactona/farmacologia , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetaminofen/análogos & derivados , Acetaminofen/metabolismo , Animais , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Bile/metabolismo , Western Blotting , Colestase Intra-Hepática/induzido quimicamente , Colestase Intra-Hepática/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/metabolismo , Glutationa/metabolismo , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Tamanho do Órgão/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SLC4A , Espironolactona/uso terapêutico , Regulação para Cima/efeitos dos fármacosRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Digoxina/farmacocinética , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Espironolactona/farmacologia , Administração Oral , Animais , Transporte Biológico/efeitos dos fármacos , Interações Medicamentosas , Masculino , Ratos , Ratos WistarRESUMO
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.
Assuntos
Ductos Biliares/metabolismo , Colestase/metabolismo , Dinitroclorobenzeno/farmacocinética , Glutationa/análogos & derivados , Rim/metabolismo , Animais , Membrana Celular/metabolismo , Glutationa/biossíntese , Glutationa/sangue , Glutationa/urina , Glutationa Transferase/metabolismo , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Córtex Renal/metabolismo , Ligadura , Fígado/metabolismo , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ratos , Ratos WistarRESUMO
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.
Assuntos
Etinilestradiol/farmacologia , Fígado/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetaminofen/análogos & derivados , Acetaminofen/análise , Acetaminofen/metabolismo , Animais , Bile/efeitos dos fármacos , Bile/metabolismo , Fígado/metabolismo , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Perfusão , Ratos , Ratos Wistar , Fatores de Tempo , Regulação para CimaRESUMO
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.
Assuntos
Bile/efeitos dos fármacos , Colestase/prevenção & controle , Etinilestradiol , Galactosamina/farmacologia , Fígado/efeitos dos fármacos , Animais , Bile/química , Bile/fisiologia , Colestase/induzido quimicamente , Colestase/metabolismo , Etinilestradiol/análogos & derivados , Etinilestradiol/análise , Etinilestradiol/metabolismo , Etinilestradiol/toxicidade , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Ácido Tauroquenodesoxicólico/metabolismo , Uridina/farmacologia , Uridina Difosfato Ácido Glucurônico/antagonistas & inibidores , Uridina Difosfato Ácido Glucurônico/metabolismoRESUMO
Despite its toxicity, acetaminophen (APAP) is used increasingly as an analgesic, antipyretic, and anti-inflammatory agent. We examined the effect of prior exposure to APAP on its biliary and urinary elimination. The biliary and urinary elimination of a test dose of APAP (150 mg/kg i.v.) was determined in male Wistar rats 24 h after pretreatment with vehicle, a single dose (1.0 g/kg i.p.), or increasing daily doses (0.2, 0.3, 0.6, and 1.0 g/kg/day i.p.) of APAP. Although elimination of the parent APAP was minimally affected, biliary excretion of APAP glucuronide was significantly decreased 70 and 80%, whereas urinary excretion was significantly increased 90 and 100% in the groups pretreated with single and repeated doses of APAP, respectively, relative to vehicle controls. Western analysis and confocal immunofluorescent microscopy indicated a marked increase in hepatic expression of multidrug resistance-associated protein 3 (Mrp3) in both groups pretreated with APAP, relative to expression of Mrp2. ATP-dependent transport of [3H]taurocholate, an Mrp3 substrate, was significantly increased in basolateral liver plasma membrane vesicles from rats pretreated with repeated doses of APAP relative to controls. Enterohepatic recirculation of APAP glucuronide after administration of the same test dose of the drug was significantly decreased in rats pretreated with repeated doses of APAP. These data indicate that APAP pretreatment induced a shift from biliary to urinary elimination of APAP glucuronide, consistent with the increased expression of Mrp3 in the basolateral domain of the hepatocyte. We postulate that decreased enterohepatic recirculation contributes to decreased APAP hepatotoxicity by reducing liver exposure.
Assuntos
Acetaminofen/análogos & derivados , Acetaminofen/farmacologia , Acetaminofen/urina , Analgésicos não Narcóticos/farmacologia , Sistema Biliar/metabolismo , Acetaminofen/metabolismo , Acetaminofen/farmacocinética , Analgésicos não Narcóticos/farmacocinética , Animais , Western Blotting , Relação Dose-Resposta a Droga , Masculino , Microscopia Confocal , Ratos , Ratos WistarRESUMO
Ethinylestradiol (EE) induces cholestasis by affecting bile salt-dependent and -independent fractions of the bile flow. The decrease in bile salt-independent flow is thought to be due, in part, to a reduction in the expression of the canalicular transporter Mrp2. The impact of modulation of Mrp2 function by sodium ursodeoxycholate (UDC) in EE cholestasis is unknown. We evaluated the protective effect of UDC on EE-induced impairment of Mrp2 activity in vivo and in isolated hepatocytes, by using the substrate dinitrophenyl S-glutathione (DNP-SG). EE was administered to male Wistar rats at a dose of 5 mg/kg s.c. for 5 days. UDC was coadministered with EE at a dose of 25 mg/kg b.wt. i.p. for the same period. EE alone reduced DNP-SG biliary excretion by 55% when compared with controls. Coadministration with UDC partially restored the alteration. Secretion rate of DNP-SG was decreased by 30% in isolated hepatocytes from EE-treated rats, but, contrary to in vivo results, UDC coadministration did not restore DNP-SG transport, likely as a consequence of bile salt washout resulting from the isolation procedure. As a confirmation, tauroursodeoxycholate hepatocyte preloading significantly increased Mrp2 activity. Western blotting analysis of Mrp2 indicated that EE administration significantly reduced its level in total and plasma membranes and that UDC coadministration failed to revert this alteration. In conclusion, UDC improvement in Mrp2 transport activity in vivo likely derived from a direct enhancement of Mrp2 function rather than from a restoration of its expression levels. This provides a novel mechanism explaining the beneficial effects of UDC in EE-induced cholestasis.