María L. Ruiz

Ca2+-dependent protein kinase C isoforms are critical to estradiol 17β-D-glucuronide–induced cholestasis in the rat†

The endogenous estradiol metabolite estradiol 17β‐D‐glucuronide (E217G) induces an acute cholestasis in rat liver coincident with retrieval of the canalicular transporters bile salt export pump (Bsep, Abcc11) and multidrug resistance‐associated protein 2 (Mrp2, Abcc2) and their associated loss of function. We assessed the participation of Ca2+‐dependent protein kinase C isoforms (cPKC) in the cholestatic manifestations of E217G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHCs). In PRL, E217G (2 μmol/liver; intraportal, single injection) maximally decreased bile flow, total glutathione, and [3H] taurocholate excretion by 61%, 62%, and 79%, respectively; incorporation of the specific cPKC inhibitor Gö6976 (500 nM) in the perfusate almost totally prevented these decreases. In dose‐response studies using IRHC, E217G (3.75–800 μM) decreased the canalicular vacuolar accumulation of the Bsep substrate cholyl‐lysylfluorescein with an IC50 of 54.9 ± 7.9 μM. Gö6976 (1 μM) increased the IC50 to 178.4 ± 23.1 μM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Mrp2 substrate, glutathione methylfluorescein. Prevention of these changes by Gö6976 coincided with complete protection against E217G‐induced retrieval of Bsep and Mrp2 from the canalicular membrane, as detected both in the PRL and IRHC. E217G also increased paracellular permeability in IRHC, which was only partially prevented by Gö6976. The cPKC isoform PKCα, but not the Ca2+‐independent PKC isoform, PKCϵ, translocated to the plasma membrane after E217G administration in primary cultured rat hepatocytes; Gö6976 completely prevented this translocation, thus indicating specific activation of cPKC. This is consistent with increased autophosphorylation of cPKC by E217G, as detected via western blotting. Conclusion: Our findings support a central role for cPKC isoforms in E217G‐induced cholestasis, by inducing both transporter retrieval from the canalicular membrane and opening of the paracellular route. (HEPATOLOGY 2008;48:1885‐1895.)

Pregnane X receptor mediates the induction of P-glycoprotein by spironolactone in HepG2 cells

We evaluated the effect of spironolactone (SL), a well-known inducer of biotransformation and elimination pathways, on the expression and activity of P-glycoprotein (P-gp/ABCB1/MDR1), a major xenobiotic transporter, in HepG2 cells, as well as the potential mediation of pregnane X nuclear receptor (PXR). Cells were exposed to SL (1, 5, 10, 20 or 50 μM) for 48 h. Expression of P-gp and its mRNA levels were estimated by Western blotting and real time PCR, respectively. P-gp activity was inversely correlated with the ability of the cells to accumulate the model substrate rhodamine 123 (Rh123, 5 μM), in the presence or absence of verapamil (50 μM), a P-gp inhibitor. At the highest dose of SL tested, P-gp and MDR1 mRNA levels were significantly increased (73 and 108%) with respect to control cells. Rh123 accumulation was concomitantly reduced and verapamil was able to abolish this effect, confirming P-gp participation. Additionally, we tested the cytotoxicity of doxorubicin, a model substrate of P-gp, under inducing conditions. HepG2 cells treated with SL exhibited higher viability, i.e. less doxorubicin toxicity, than control cells, consistent with P-gp up-regulation. When HepG2 cells were treated with SL in the presence of ketoconazole (KTZ), a non-specific nuclear receptor inhibitor, the up-regulation of P-gp was suppressed. To further identify the nuclear receptor involved, cells were transfected with a siRNA directed against human PXR, leading to a 74% decrease in PXR protein levels, which totally abolished SL induction of P-gp. We conclude that SL up-regulates P-gp expression, likely at transcriptional level, and its efflux activity in HepG2 cells. This effect is mediated by PXR. Thus, ligands of PXR such as SL may alter the disposition and toxicity of other xenobiotics, including drugs of therapeutic use, that are P-gp substrates.

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.

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

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

Expression and function of Oat1 and Oat3 in rat kidney exposed to mercuric chloride

This study was designed to evaluate the expression and function of the organic anion transporters, Oat1 and Oat3, in rats exposed to a nephrotoxic dose of HgCl2. Oat1 protein expression increased in renal homogenates and decreased in renal basolateral membranes from HgCl2 rats, while Oat3 protein abundance decreased in both kidney homogenates and basolateral membranes. The lower protein levels of Oat1 and Oat3 in basolateral membranes explain the lower uptake capacity for p-aminohippurate (in vitro assays) and the diminution of the systemic clearance of this organic anion (in vivo studies) observed in treated rats. Since both transporters mediate mercury access to the renal cells, their down-regulation in basolateral membranes might be a defensive mechanism developed by the cell to protect itself against mercury injury. The pharmacological modulation of the expression and/or the function of Oat1 and Oat3 might be an effective therapeutic strategy for reducing the nephrotoxicity of mercury.

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 17α-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 μmol/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.

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 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.

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.

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 μmol/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.

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

The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for the multidrug resistance-associated proteins (Mrps), was assessed in bile duct-ligated (BDL) rats 1, 7, and 14 days after surgery, using an in vivo perfused jejunum model with simultaneous urine collection. A single i.v. dose of 30 μmol/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 γ-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 α, μ, and π, 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.