Hepatobiliary excretion of acetaminophen glutathione conjugate and its derivatives in transport-deficient (TR-) hyperbilirubinemic rats.

The involvement of the canalicular multidrug resistance protein 2 (Mrp2) in the hepatobiliary excretion of acetaminophen (APAP)-glutathione (GSH) conjugate and its derivatives was investigated using transport-deficient (TR- rats. Although no differences in the biliary concentration of APAP itself were detected between normal Wistar and TR- rats, significant differences in the biliary disposition of several conjugated metabolites of APAP were detected. APAP-GSH was virtually absent in bile from TR- rats. Also, biliary concentrations of APAP-mercapturate (NAC; N-acetylated l-cysteine) and APAP-GLU were significantly reduced in TR- rats. No differences in the biliary concentration of APAP-cysteinylglycine/cysteine (CG/CYS) were detected between normal and mutant rats. The cumulative amounts of APAP-CG/CYS and APAP-NAC excreted in urine of mutant rats were decreased, whereas APAP-GLU was markedly increased. Analysis of liver samples revealed that APAP-GSH and APAP-NAC accumulate in mutant rat livers. Our results support the direct involvement of Mrp2 in the hepatobiliary excretion of several conjugated metabolites of APAP, including APAP-GSH and APAP-NAC, and provide relevant information on processes that may be involved with both their hepatic basolateral transport and renal elimination.     

Effect of acetaminophen on expression and activity of rat liver multidrug resistance-associated protein 2 and P-glycoprotein

We evaluated the effect of acetaminophen (APAP), given as a single, 1g/kg body weight dose, on expression and activity of rat liver multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein (P-gp), two major canalicular drug transporters. The studies were performed 24h after administration of the drug. APAP induced an increase in plasma membrane content of Mrp2 detected by western blotting, consistent with increased detection of the protein at the canalicular level by immunoflourescence microscopy. In vivo biliary excretion of dinitrophenyl-S-glutathione, a well known Mrp2 substrate, was slightly but significantly increased by APAP, agreeing well with upregulation of the transporter. Basal biliary excretion of oxidized glutathione, an endogenous Mrp2 substrate, was also increased by APAP, likely indicating increased hepatic synthesis as a result of APAP-induced oxidative stress followed by accelerated canalicular secretion mediated by Mrp2. APAP also increased the expression of P-gp detected by western blotting and immunofluorescence microscopy as well as the in vivo biliary secretory rate of digoxin, a model P-gp substrate. Because specific APAP-conjugated metabolites are Mrp2 substrates, we postulate that induction of Mrp2 by APAP may represent an adaptive mechanism to accelerate liver disposition of the drug. In addition, increased Mrp2-mediated elimination of oxidized glutathione may be essential in maintaining the redox equilibrium in the hepatocyte under conditions of APAP-induced oxidative stress.     

Effects of glycyrrhizin on biliary transport and hepatic levels of glutathione in rats

The purpose of the current study was to determine whether glycyrrhizin (GL) maintains hepatic glutathione (GSH) levels by inhibiting GSH biliary secretion in normal rats. The effects of glycyrrhizin on hepatic glutathione content, bile flow and biliary secretion of glutathione were examined. Because glutathione is a substrate for multidrug resistance associated protein‐2 (Mrp2/ABCC2), the inhibitory effects of GL on Mrp2 in isolated perfused rat liver and in Mrp2‐expressing Sf9 membrane vesicles were also examined using the Mrp2 substrate methotrexate (MTX) and estradiol‐17‐β‐glucuronide (E₂17G). The hepatic content of glutathione in rats following GL perfusion (43.7 µmol/l) in isolated liver perfusion and GL intravenous treatment (25 mg/kg) was significantly higher than that for the control. A marked and dose‐dependent decrease in the excretion of glutathione was observed. In addition, the secretion rate of MTX was decreased by 57% in isolated liver perfusion in GL‐treated rats. Moreover the ATP‐dependent uptake of E₂17G by Mrp2 membrane vesicles was decreased by 75.9% in the 20 µm GL group and by 60.5% in the 2 µm GL group. In conclusion, glycyrrhizin increases hepatic glutathione content possibly through inhibition of Mrp2 which then reduces the biliary excretion of glutathione. Copyright © 2012 John Wiley & Sons, Ltd.     

Transport deficient (TR-) hyperbilirubinemic rats are resistant to acetaminophen hepatotoxicity

The biliary excretion of acetaminophen (APAP) is reduced in transport deficient (TR⁻) hyperbilirubinemic rats lacking the multidrug resistance-associated protein 2 (Mrp2). This mutant strain of Wistar rats has impaired biliary excretion of organic anions and increased hepatic glutathione. The rational for this study was to determine if there is an altered risk for liver damage by APAP in the absence of Mrp2. Therefore, the susceptibility of TR⁻ rats to APAP hepatotoxicity was investigated. Male Wistar and TR⁻ rats were fasted overnight before APAP treatment (1 g/kg). Hepatotoxicity was assessed 24 h later by plasma sorbitol dehydrogenase activity and histopathology. In other studies, TR⁻ rats received buthionine sulfoximine before APAP to reduce hepatic glutathione to values similar to those in Wistar rats. mRNA expression of APAP metabolizing enzymes was also measured in naïve animals. Wistar rats treated with APAP showed significant elevations in plasma sorbitol dehydrogenase activity, while no increases in enzyme activity were observed in TR⁻ rats. Histopathology was in agreement. Hepatic non-protein sulfhydryls were significantly lower in Wistar rats receiving APAP than in TR⁻ rats. TR⁻ rats treated with buthionine sulfoximine and APAP showed dramatic increases in hepatotoxicity. TR⁻ rats had increased mRNA expression of several APAP metabolizing enzymes. Mrp2 expression not only is important in biliary excretion, but also influences the toxic potential of reactive intermediates by controlling intrahepatic GSH and possibly drug metabolism.     

Mechanisms of impaired biliary excretion of acetaminophen glucuronide after acute phenobarbital treatment or phenobarbital pretreatment.

Previous studies have demonstrated that phenobarbital (PB) significantly impairs the biliary excretion of acetaminophen glucuronide (AG) in rats. Studies also suggested that Mrp2 mediates AG biliary excretion, and Mrp3 is involved in AG basolateral export. It was hypothesized that inhibition of Mrp2-mediated AG transport by PB or PB metabolites, and PB induction of Mrp3, may contribute to the impaired biliary excretion of AG by PB. In the present study, the hepatobiliary transport of AG in single-pass isolated perfused Wistar and TR(-) rat livers was investigated. The AG biliary clearance was markedly decreased, and the AG basolateral clearance was significantly increased in TR(-) rat livers. Uptake of AG by Mrp2 and Mrp3, and inhibition of Mrp2- and Mrp3-mediated transport by PB and major PB metabolites, were investigated with rat Mrp2- or Mrp3-expressing Sf9 cell plasma membrane vesicles (Sf9-PMVs). AG was transported by Mrp3 (K(m) approximately 0.91 mM). Net ATP-dependent AG uptake into Mrp2-expressing Sf9-PMVs could not be detected directly. However, AG significantly inhibited Mrp2-mediated 5-(and 6)-carboxy-2',7'-dichlorofluorescein (CDF) transport. p-Hydroxyphenobarbital glucuronide (p-OHPBG), but not PB or p-hydroxyphenobarbital, significantly inhibited Mrp2-mediated CDF transport. The IC(50) values for p-OHPBG inhibition of Mrp2-mediated CDF uptake and Mrp3-mediated AG transport were similar (approximately 0.68 and 0.46 mM, respectively). PB treatment (80 mg/kg/day x 4 days) markedly increased hepatic Mrp3 expression in Wistar rats. In conclusion, inhibition of Mrp2-mediated AG transport by p-OHPBG provided one possible explanation for the impaired biliary excretion of AG after acute PB treatment. However, impaired biliary excretion of AG after PB pretreatment may be attributed primarily to the induction of hepatic Mrp3 by PB.     

Efflux transporter expression and acetaminophen metabolite excretion are altered in rodent models of nonalcoholic fatty liver disease.

Efflux transporters are responsible for the excretion of numerous xenobiotics and endobiotics and thus play an essential role in proper liver and kidney function. Nonalcoholic fatty liver diseases (NAFLDs) comprise a spectrum of disorders that range from simple fatty liver (SFL) to nonalcoholic steatohepatitis (NASH). Although the precise events leading to NAFLD are unclear, even less is known about the effects on efflux transporter expression and drug disposition. The purpose of this study was to determine the effect of NAFLD on efflux transporter expression in rat liver as well as on acetaminophen (APAP) metabolite excretion. To simulate SFL and NASH, rats were fed either a high-fat (HF) or a methionine- and choline-deficient (MCD) diet for 8 weeks. In the livers of MCD rats, there were striking increases in both mRNA and protein levels of multidrug resistance-associated protein (Mrp) 3, Mrp4, and breast cancer resistance protein, as well as increased Mrp2 protein. After administration of a nontoxic dose of APAP, biliary concentrations of APAP-sulfate, APAP-glucuronide (APAP-GLUC), and APAP-glutathione were reduced in MCD rats. The effects of the HF diet on both transporter expression and APAP disposition were by comparison far less dramatic than the MCD diet-induced alterations. Whereas APAP-sulfate levels were also decreased in MCD rat plasma, the levels of the Mrp3 substrate APAP-GLUC were elevated. Urinary elimination of APAP metabolites was identical between groups, except for APAP-GLUC, the concentration of which was 80% higher in MCD rats. These studies correlate increased hepatic Mrp3 protein in the MCD model of NASH with increased urinary elimination of APAP-GLUC. Furthermore, the proportional shift in elimination of APAP metabolites from bile to urine indicates that MCD-induced alterations in efflux transporter expression can affect the route of drug elimination.     

Effects of clofibrate and indocyanine green on the hepatobiliary disposition of acetaminophen and its metabolites in male CD-1 mice

1. The effects of clofibrate (CFB) and indocyanine green (ICG) on the biliary excretion of acetaminophen (APAP) and its metabolites were investigated. 2. Male CD-1 mice were pretreated with 500?mg CFB/kg, i.p. for 10 days. Controls received corn oil vehicle only. After overnight fasting, common bile duct-cannulated mice were challenged with a non-toxic dose of APAP (1 mmol/kg, i.v.). 3. CFB pretreatment did not affect bile flow rate, nor did it affect the cumulative biliary excretion of APAP and its conjugated metabolites. 4. Additional CFB or corn oil pretreated mice were given 30 μmol indocyanine green (ICG)/kg, i.v., immediately before APAP dosing. ICG is a non-metabolizable organic anion that is completely excreted into the bile through a canalicular transport process for organic anions. 5. ICG significantly decreased the bile flow rate and biliary concentration of APAPglutathione, APAP-glucuronide and APAP-mercapturate within the first hour after dosing without affecting the biliary concentration of APAP. 6. The results indicate thatCFB pretreatment does not affect the total amount of APAP and its metabolites excreted in bile. They also suggest that the biliary excretion of several conjugated metabolites of APAP share the same excretory pathway with the organic anion ICG.     

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.     

Therapeutic potential of Moringa oleifera extracts against acetaminophen-induced hepatotoxicity in rats

Context: Moringa oleifera Lam. (Moringaceae) is a rich source of essential minerals and antioxidants; it has been used in human and animal nutrition. The leaves and flowers are being used by the population with great dietary importance.

Objective: The present study was to investigate the therapeutic effects of the hydroethanolic extract of Moringa oleifera (MO) leaves and flowers against hepatotoxicity induced by acetaminophen (APAP) in rats.

Materials and methods: In the hepatoprotective study, either flowers or leaves of hydroethanolic extract (200 or 400?mg/kg bw through IP injection) were administered an hour after APAP administration. N-Acetylcysteine (NAC) was used as the positive control for this study. Liver and kidney function tests including lipid peroxidation levels were analyzed and histopathological changes of liver and kidney were also observed.

Results: Acetaminophen-induced hepatotoxicity increased the activities of liver marker enzymes. Histologically, the liver was observed to have inflammation and bridging necrosis. Liver marker enzymes were significantly reduced when treated with flower and leaf extracts of MO in animals with APAP induced toxicity. In addition, there were no significant changes observed in clinical markers of kidney function. Histological observation on liver tissue from the rats treated with MO flower and leaf extract showed reduction in the severity of the liver damage.

Discussion and conclusion: These results indicated the possible therapeutic action of flower and leaf extract from MO in protecting liver damage in rats given an over dosage of APAP.     

The important role of Bcrp (Abcg2) in the biliary excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in mice

The role of Mrp2, Bcrp, and P-glycoprotein in the biliary excretion of acetaminophen sulfate (AS) and glucuronide (AG), 4-methylumbelliferyl sulfate (4MUS) and glucuronide (4MUG), and harmol sulfate (HS) and glucuronide (HG) was studied in Abcc2(-/-), Abcg2(-/-), and Abcb1a(-/-)/Abcb1b(-/-) mouse livers perfused with the respective parent compounds using a cassette dosing approach. Biliary clearance of the sulfate conjugates was significantly decreased in Bcrp-deficient mouse livers, resulting in negligible biliary excretion of AS, 4MUS, and HS. It is noteworthy that the most profound decrease in the biliary clearance of the glucuronide conjugates was observed in Bcrp-deficient mouse livers, although the biliary clearance of 4MUG was also approximately 35% lower in Mrp2-deficient mouse livers. As expected, biliary excretion of conjugates was not impaired in P-glycoprotein-deficient livers. An appreciable increase in perfusate recovery due to a shift in the directionality of metabolite excretion, from bile to perfusate, was noted in knockout mice only for conjugates whose biliary clearance constituted an appreciable (> or =37%) fraction of total hepatic excretory clearance (i.e., 4MUS, HG, and HS). Biliary clearance of AG, AS, and 4MUG constituted a small fraction of total hepatic excretory clearance, so an appreciable increase in perfusate recovery of these metabolites was not observed in knockout mice despite markedly decreased biliary excretion. Unlike in rats, where sulfate and glucuronide conjugates were excreted into bile predominantly by Mrp2, mouse Bcrp mediated the biliary excretion of sulfate metabolites and also played a major role in the biliary excretion of the glucuronide metabolites, with some minor contribution from mouse 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 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.     

Metabolism and disposition of gemfibrozil in Wistar and multidrug resistance-associated protein 2-deficient TR− rats

1. The roles of multidrug resistance-associated protein (Mrp) 2 deficiency and Mrp3 up-regulation were evaluated on the metabolism and disposition of gemfibrozil.

2. Results from in vitro studies in microsomes showed that the hepatic intrinsic clearance (CL_int) for the oxidative metabolism of gemfibrozil was slightly higher (1.5-fold) in male TR^? rats, which are deficient in Mrp2, than in wild-type Wistar rats, whereas CL_int for glucuronidation was similar in both strains.

3. The biliary excretion of intravenously administered [¹⁴C]gemfibrozil was significantly impaired in TR^? rats compared with Wistar rats (22 versus 93% of the dose excreted as the acyl glucuronides over 72?h). Additionally, the extent of urinary excretion of radioactivity was much higher in TR^? than in Wistar rats (78 versus 2.6% of the dose).

4. There were complex time-dependent changes in the total radioactivity levels and metabolite profiles in plasma, liver and kidney, some of which appeared to be related to the up-regulation of Mrp3.

5. Overall, it was demonstrated that alterations in the expression of the transporters Mrp2 and Mrp3 significantly affected the excretion as well as the secondary metabolism and distribution of [¹⁴C]gemfibrozil.     

Thearubigins protect against acetaminophen-induced hepatic and renal injury in mice: biochemical,histopathological, immunohistochemical,and flow cytometry study

Context: Acetaminophen toxicity is used as a model for studying chemical toxicity. N-acetylcysteine (NAC) is used for the treatment of hepatotoxicity; however, there is no specific therapy for nephrotoxicity. Objective: This study was designed to investigate the potential protective effect of black tea extract (BTE) and its main phenolic pigment, thearubigins (TRs), against acetaminophen (APAP)-induced hepatic and renal injury in mice. Materials and methods: Besides control groups, six groups (n?=?8) were given intraperitoneally APAP (300?mg/kg) and then after 1.5 hours were treated intraperitoneally as follows: NAC (318?mg/kg), BTE (3%, 4.5%), and TRs (50, 60, and 70?mg/kg). Six hours post-APAP injection, blood was collected for biochemical measurements. Later, liver and kidneys were removed for histopathological, immunohistochemical, and flow cytometry studies. Results: APAP increased alanine aminotransferase and malondialdehyde and decreased glutathione levels in blood. Treatments significantly reversed these changes mostly with NAC and TRs70. TRs showed dose-dependent significant differences. The APAP-induced central lobular hepatic necrosis and increased TUNEL positivity were mild with co-administration of NAC and TRs (60,?70) while moderate with co-administration of BTE (3,?4.5) and TRs50. The APAP-increased serum creatinine level was significantly reversed by treatments (mostly TRs60,?70). The APAP-induced renal tubular epithelial degeneration and necrosis were mild with co-administration of TRs (60,?70) while moderate with co-administration of NAC, BTE (3,?4.5), and TRs50. The APAP-accumulated apoptotic cells in sub-G₁ phase were significantly decreased by treatments, mostly by NAC and TRs70 in the liver and TRs (60,?70) in kidneys. Conclusion: Thearubigins protected against acetaminophen-induced hepatotoxicity and nephrotoxicity in mice possibly through their antioxidant activity.     

Therapeutic effect of liposomal-N-acetylcysteine against acetaminophen-induced hepatotoxicity

Abstract

Background: Acetaminophen (APAP) is an antipyretic analgesic drug that when taken in overdose causes depletion of glutathione (GSH) and hepatotoxicity. N-acetylcysteine (NAC) is the antidote of choice for the treatment of APAP toxicity; however, due to its short-half-life repeated dosing of NAC is required.

Purpose: To determine whether a NAC-loaded liposomal formulation (Lipo-NAC) is more effective than the conventional NAC in protecting against acute APAP-induced hepatotoxicity.

Methods: Male Sprague–Dawley rats were challenged with an intragastric dose of APAP (850?mg/kg b.wt.); 4?h later, animals were administered saline, NAC, Lipo-NAC or empty liposomes and sacrificed 24?h post-APAP treatment.

Results: APAP administration resulted in hepatic injury as evidenced by increases in plasma bilirubin, alanine (AST) and aspartate (ALT) aminotransferase levels and tissue levels of lipid peroxidation and myeloperoxidase as well as decreases in hepatic levels of reduced GSH, GSH peroxidase and GSH reductase. Treatment of animals with Lipo-NAC was significantly more effective than free NAC in reducing APAP-induced hepatotoxicity. Histological evaluation showed that APAP caused periacinar hepatocellular apoptosis and/or necrosis of hepatocytes around the terminal hepatic venules which was reduced by NAC treatment, the degree of reduction being greater for Lipo-NAC.

Conclusion: These data suggest that administration of Lipo-NAC ameliorated the APAP-induced hepatotoxicity.     

The Mouse Liver Slice System as an In Vitro Model for Testing the Protective Effect of N-Acetylcysteine Against Hepatotoxic Agents

Abstract

Summary: An in vitro liver slice system was used to test the antidotal effect of N-acetylcysteine (NAC) against hepatotoxic agents. Incubation of mouse slices with acetaminophen (APAP) in a concentration of 6.8 mg/ml resulted in lactate dehydrogenase (LDH) leakage from the cells. NAC protected the tissue from APAP-induced hepatocellular damage in a dose-dependent manner. Maximal protection was achieved by 10 mM of the sulfhydryl compound when applied 20 min following APAP intoxication. Longer intervals, concurrent administration, or preincubation of the slices with the SH reagent were less protective against the hepatotoxin. The liver slice system has been used to test NAC antidotal activity against other hepatotoxins known to be metabolized to reactive radicals, such as carbon tetrachloride (CC1₄), bromobenzene, and furosemide. The hepatocellular damage caused by these agents was slightly reduced by the sulfhydryl agent. These findings may demonstrate the ability of the in vitro liver slice system to serve as a tool for primary assessment of the activity of various antidotes against toxic agents.     

Hearing,reactive metabolite formation,and oxidative stress in cochleae after a single acute overdose of acetaminophen: an in vivo study

Context: Although the liver is the primary target organ in acetaminophen (APAP) toxicity, other organs are affected. Previous data suggested that chronic APAP abuse can be ototoxic and the mechanism involves APAP-induced oxidative stress and reactive metabolite (N-acetyl-p-benzoquinone imine, NAPQI)-induced endoplasmic reticulum stress. However, the effect of a single acute overdose on hearing has not been tested.

Objectives: To determine if a single acute APAP overdose causes hearing damage, and to explore possible mechanisms of APAP ototoxicity.

Materials and methods: Male C57BL/6?J mice were treated with a single human-relevant overdose of APAP (300?mg APAP per kg bodyweight). Blood, liver and cochleae were harvested at 0, 2, 6 and 24?h post-APAP. In some mice, auditory brainstem responses (ABRs) to a range of frequencies were measured at 24?h. The furosemide plus kanamycin (FS/K) model of drug ototoxicity was used as a positive control for hearing loss. NAPQI formation after APAP was assessed by measuring glutathione depletion and covalent protein binding, and oxidative stress was assessed by measuring glutathione disulfide.

Results: There was no evidence of reactive metabolite formation or hearing loss after a single overdose of APAP at a clinically relevant dose. However, there was a transient increase in oxidative stress.

Discussion: Although a single acute overdose was not ototoxic, there was evidence of oxidative stress which may support a role for oxidative stress in hearing loss due to chronic APAP abuse.

Conclusion: A single human-relevant acute overdose of APAP causes transient oxidative stress in cochleae but not hearing loss.     

A membrane vesicle-based assay to enable prediction of human biliary excretion

Abstract

1. Prediction of biliary excretion is a challenge due to the lack of in vitro assays. Our laboratory previously demonstrated a highly significant correlation between in vitro IC₅₀ values against mrp2 using rat canalicular liver plasma membrane vesicles and in vivo biliary excretion (Colombo et al., 2012). This study explores the possibility of predicting in vivo biliary excretion in human using membrane vesicles prepared from MDCKII cells transfected with human ABCC2.

2. In vitro MRP2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) in inside-out membrane vesicles isolated from MDCK-ABCC2 cells. CDCF uptake was time- and concentration-dependent (K_m of 4.0?±?1.2?µM and a V_max of 7.8?±?0.9?pmol/mg/min) and inhibited by benzbromarone and MK-571 with IC₅₀ values of 1.2 and 7.6?µM, respectively.

3. A significant linear correlation (r^2?=?0.790) between the in vitro IC₅₀ values from the described MRP2 assay and in vivo biliary excretion in humans was observed using 11 well-documented drugs covering low to high biliary excretions.

4. This study demonstrates, for the first time, that inhibition of CDCF uptake in MDCKII-ABCC2 vesicles not only provides a screening assay to assess MRP2 drug–drug interaction potential, but is also predictive of human MRP2-mediated biliary excretion.