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.Fil: Rigalli, Juan Pablo. Universität Heidelberg; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Perdomo, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Arias, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Theile, Dirk. Universität Heidelberg; AlemaniaFil: Weiss, Johanna. Universität Heidelberg; AlemaniaFil: Mottino, Aldo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Catania, Viviana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

Hormonal Modulation of Hepatic cAMP Prevents Estradiol 17β-d-Glucuronide-Induced Cholestasis in Perfused Rat Liver

BACKGROUND: Estradiol-17β-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.Fil: Zucchetti, Andrés Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Barosso, Ismael Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Boaglio, Andrea Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Roma, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Crocenzi, Fernando Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Sanchez Pozzi, Enrique Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentin

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.Fil: Novak, Analia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; ArgentinaFil: Delli Carpini, Griselda. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; ArgentinaFil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Rubio, Modesto Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas (i); ArgentinaFil: Mottino, Aldo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Ghanem, Carolina Inés. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas (i); Argentin

Modulation of Biotransformation Systems and ABC Transporters by Benznidazole in Rats

The effect of antichagasic benznidazole (BZL; 100mg/kg body weight/day, 3 consecutive days, intraperitoneally) on biotransfor-mation systems and ABC transporters was evaluated in rats. Expression of cytochrome P-450 (CYP3A), UDP-glucuronosyltrans-ferase (UGT1A), glutathioneS-transferases (alpha glutathioneS-transferase [GST-], GST-, andGST-), multidrug-resis-tance-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%) andGST-(40%) and in intestinal GST-(72% in jejunumand 136% in ileum) were detected. Significant increases in Mrp2 (300%) and P-gp (500%) proteins in liver from BZL-treated rats were observedwithout 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 correlatedwell with the upregulation of proteins in liver and jejunum. Plasma decay of a test dose of BZL (5mg/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 BZLwas higher (60%) in the BZL group, and urinary excretion of BZL did not showdifferences between groups. The amount of absorbed BZL in intestinal sacs was lower (25%) in pretreated rats than in controls. In conclusion, induction of biotransforma-tion enzymes and/or transporters by BZL could increase the clearance and/or decrease the intestinal absorption of coadminis-tered drugs that are substrates of these systems, including BZL itself.Fil: Perdomo, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Rigalli, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Echenique, Claudia G.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Catania, Viviana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentin

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.Fil: Ceré, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Sedlmeier, María Guillermina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Semeniuk, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Frances, Daniel Eleazar Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Ronco, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Rigalli, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Catania, Viviana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentin

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 0.6 g/kg, i.p.) of APAP on enterohepatic recirculation and liver toxicity of a subsequent i.p. toxic dose of 1 g/kg, given 24 h 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 6 h 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.Fil: Ghanem, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Llesuy, Susana Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; ArgentinaFil: Catania, Viviana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Bengochea, Laura Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Mottino, Aldo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentin

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

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.Fil: Arana, Maite Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Tocchetti, Guillermo Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Domizi, Pablo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Arias, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Rigalli, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Banchio, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mottino, Aldo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentin

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.Fil: Rigalli, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Ciriaci, Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Arias, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Ceballos Mancini, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Mottino, Aldo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Ghanem, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas (i); ArgentinaFil: Catania, Viviana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentin

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.Fil: Semeniuk, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Ceré, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Ciriaci, Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Bucci Muñoz, Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Quiroga, Ariel Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Luquita, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Roma, Stella Maris. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas. Cátedra de Histología y Embriología; ArgentinaFil: Catania, Viviana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Mottino, Aldo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Rigalli, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentin