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

doi:10.1016/j.bcp.2005.09.024

Biochemical Pharmacology

Volume 70, Issue 12, 5 December 2005, Pages 1832-1839

https://doi.org/10.1016/j.bcp.2005.09.024 Get rights and content

Abstract

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.

Introduction

Acetaminophen (APAP) and its conjugated metabolites can be found in urine and bile of mice and rats. A considerable amount of APAP is excreted in bile, mainly as the glutathione (APAP-GSH) and glucuronide (APAP-GLUC) conjugates [1], [2]. Transport mechanisms for the hepatobiliary excretion of APAP and its metabolites are not completely understood. Recent studies indicate that the multidrug resistance-associated proteins 2 and 3 (Mrp2 and 3, ABCC2 and ABCC3, respectively) are involved in this process. The biliary excretion of APAP-GLUC and APAP-sulfate was reduced in isolated perfused livers from transport deficient (TR⁻) rats [3]. This mutant strain of Wistar rats lacks expression of functional Mrp2 [4], [5].

Mrp2 is involved in the biliary excretion of amphiphilic organic anions including non-bile acid organic anions, glucuronide and glutathione conjugates [6], [7], [8], [9]. Excretion of these compounds into bile is impaired in TR⁻ rats [10], [11]. Our laboratory demonstrated that TR⁻ rats receiving APAP have decreased biliary excretion of APAP-GSH, APAP-GLUC and APAP-N-acetylcysteine [1]. Our studies also showed that Mrp2 deficiency results in increased urinary excretion of APAP-GLUC in TR⁻ rats. This is most likely due to compensatory up-regulation of the basolateral efflux transporter Mrp3 in TR⁻ rats [12]. Studies in Mrp3^−/− mice conclusively demonstrated that the sinusoidal efflux transport of APAP-GLUC is highly dependent on Mrp3 function [13].

A key question not previously addressed is whether there is an altered risk for hepatic damage by APAP in the absence of Mrp2. Since the organic anion indocyanine green (ICG) not only produces changes in the biliary disposition of APAP similar to those seen in TR⁻ rats [14], but also has a protective effect against its hepatotoxicity [15], we decided to investigate the susceptibility of TR⁻ rats to APAP toxicity.

Hepatic GSH is important in the detoxification of the reactive metabolite of APAP, N-acetyl-p-benzoquinoneimine (NAPQI). GSH is transported into bile by a common transport-mediated process with organic anions [16]. TR⁻ rats have increased hepatic GSH levels, demonstrating that Mrp2 is important for GSH transport into bile [17]. Although the higher GSH content in liver should make these mutant rats more resistant to APAP toxicity, the dramatic changes in hepatobiliary disposition of APAP in these rats makes it difficult to anticipate their ultimate response to a toxic dose of APAP.

The results of the present studies show that TR⁻ rats are highly resistant to APAP toxicity. To investigate the role of higher hepatic GSH in this resistance, hepatic GSH content in TR⁻ rats was normalized to values in naïve Wistar rats with the use of the GSH-depleting agent buthionine sulfoximine (BSO) prior to APAP administration. Changes in gene expression of several APAP metabolizing enzymes between strains of rats were investigated also.

Section snippets

Reagents

4-Acetamidophenol (APAP), buthionine sulfoximine, glutathione, trichloroacetic acid, EDTA, 5,5′-dithio-bis(2-nitrobenzoic acid), trizma hydrochloride, trizma base and β-nicotinamine adenine dinucleotide (reduced form) were purchased from Sigma Chemical Co. (St. Louis, MO). All chemicals were of reagent grade or better.

Animals

Male Wistar rats were obtained from Charles River Laboratories (Wilmington, MA) and TR⁻ rats were bred in our animal facilities. The mutational status of our TR⁻ rats was

Studies on the susceptibility of TR⁻ rats to APAP hepatotoxicity

Plasma SDH activity in Wistar and mutant rats receiving Gum Arabic was negligible (Fig. 1). However, Wistar rats receiving APAP showed SDH values of 646.3 ± 164 U/ml. By contrast, plasma SDH values in TR⁻ rats were drastically lower (approximately 50 U/ml), indicating that the mutant rats are resistant to APAP hepatotoxicity. These results were in agreement with the histopathology. As depicted in Table 1, 100% of the animals treated with vehicle received a score of zero. Similarly, TR⁻ rats

Discussion

The human Dubin–Johnson syndrome is a rare autosomal recessive liver disorder characterized by chronic conjugated hyperbilirubinemia [29]. Patients with this condition have a compromised capacity for excreting conjugated bilirubin and other organic anions into bile. A highly homologous phenotype has been described for the transport-deficient (TR⁻) hyperbilirubinemic rat [30], [31].

Recent experiments in our laboratory and others documented a role for Mrp2 in the biliary disposition of APAP [1],

Acknowledgements

This study was supported in part by a grant from the National Institute of Environmental Health Sciences (ES07163) and a Multicultural Predoctoral Fellowship from the University of Connecticut to Vanessa M. Silva.

References (40)

L.T. Wong
Pathways of disposition of acetaminophen conjugates in the mouse
Toxicol Lett
(1981)
T. Ishikawa
ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane. Role of the ATP-dependent transport system for glutathione S-conjugates
J Biol Chem
(1990)
R.P. Oude Elferink et al.
The role of the canalicular multispecific organic anion transporter in the disposal of endo- and xenobiotics
Pharmacol Ther
(1994)
V.M. Silva
Changes in susceptibility to acetaminophen-induced liver injury by the organic anion indocyanine green
Food Chem Toxicol
(2001)
S.C. Lu
Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats
Hepatology
(1996)
A.M. Standeven et al.
Tissue-specific changes in glutathione and cysteine after buthionine sulfoximine treatment of rats and the potential for artifacts in thiol levels resulting from tissue preparation
Toxicol Appl Pharmacol
(1991)
C.G. Dietrich
Role of Mrp2 and GSH in intrahepatic cyclin of toxins
Toxicology
(2001)
G.L. Ellman
Tissue sulfhydryl groups
Arch Biochem Biophys
(1959)
J.E. Manautou
Clofibrate pretreatment diminishes acetaminophen's selective covalent binding and hepatotoxicity
Toxicol Appl Pharmacol
(1994)
J.E. Manautou
Repeated dosing with the peroxisome proliferator clofibrate decreases the toxicity of model hepatotoxic agents in male mice
Toxicology
(1998)

J.E. Manautou

Clofibrate pretreatment diminishes acetaminophen's selective covalent binding and hepatotoxicity

Toxicol Appl Pharmacol

(1994)

R. Drew et al.

The effects of buthionine sulphoximine (BSO) on glutathione depletion and xenobiotic biotransformation

Biochem Pharmacol

(1984)

A. Meister

Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy

Pharmacol Ther

(1991)

T. Hirohashi et al.

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3)

J Biol Chem

(1999)

D.J. Newton et al.

Determination of phase I metabolic enzyme activities in liver microsomes of Mrp2 deficient TR⁻ and EHBR rats

Life Sci

(2005)

C. Chen et al.

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

Drug Metab Dispos

(2003)

H. Xiong

Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(−) rats

J Pharmacol Exp Ther

(2000)

C.C. Paulusma

Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene

Science

(1996)

K. Ito

Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR

Am J Physiol

(1997)

T. Nishida

Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR⁻) with inherited conjugated hyperbilirubinemia

J Clin Invest

(1992)

Cited by (21)

Lack of multidrug resistance-associated protein 4 (Mrp4) alters the kinetics of acetaminophen toxicity
2019, Toxicology Reports
Citation Excerpt :
In both humans and rodents, altered expression of hepatic Mrp proteins is shown to alter APAP disposition [26,27]. Similarly, Mrp transporters knockout mice have been reported to have altered susceptibility to APAP toxicity [28–30]. Under baseline conditions, hepatic Mrp2 mRNA levels were decreased in Mrp4−/− mice compared WT mice while hepatic Mrp3 mRNA expression was slightly lower though not statistically significant (Fig. 4A).
Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver injury in humans and a common chemical model to investigate genetic determinants of susceptibility to drug-induced liver injury (DILI). Previous studies performed in our laboratory identified the efflux transporter multidrug resistance-associated protein 4 (Mrp4) as an inducible gene in the liver following toxic APAP exposure in both humans and rodents. In mice, blockade of hepatic Mrp4 induction following APAP administration increases susceptibility towards APAP hepatotoxicity. Collectively, these findings suggest that Mrp4 plays an important role in tolerance to APAP-induced liver injury. To further study the role of Mrp4 in APAP-induced hepatotoxicity, we treated 10–12 weeks old male wild type (WT, C57BL/6J) and Mrp4 knockout (Mrp4^−/−) mice with APAP (400 mg/Kg in saline, i.p.) or vehicle. Liver injury endpoints and hepatic gene expression were analyzed at 12, 24 and 48 h post-APAP injections. Unexpectedly, the kinetics of histologically measured liver damage and plasma ALT revealed that Mrp4^−/ mice had decreased ALT levels and hepatic necrosis compared to WT mice only at 12 h. Notably, hepatic non-protein sulfhydryl (NPSH) levels were increased in the APAP treated Mrp4^−/− mice at intervals less than 24 h, consistent with the capability of Mrp4 to export glutathione. Further gene expression analysis revealed that hepatic drug metabolism genes were downregulated in Mrp4^−/− mice at earlier time points post-APAP administration. However, despite significant decreases in endpoints of liver injury detected at an early time point after APAP treatment, these changes were not sustained at later time points as Mrp4^−/− mice ultimately had hepatic toxicity at levels comparable to WT mice. In conclusion, our data indicate that lack of Mrp4 by itself in mice does not alter susceptibility to APAP toxicity.
Acetaminophen inhibits intestinal P-Glycoprotein transport activity
2013, Journal of Pharmaceutical Sciences
Citation Excerpt :
Second, intracellular accumulation of AP in a human cell line was not affected after partial silencing of P-gp protein expression. Although transport of AP-conjugated metabolites has been well characterized and involves Mrps and Bcrp,32-36 identification of a transporter recognizing unmetabolized AP as a substrate, either involving cellular uptake or extrusion is lacking. Manov et al.37 hypothesized that AP behaves as a P-gp substrate because coincubation with Ver increased AP toxicity in HepG2 cells, which was attributed to increased AP intracellular accumulation.
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. V_max 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.
Synergistic influence of Abcb1 and Abcc2 on disposition and sterol lowering effects of ezetimibe in rats
2010, Journal of Pharmaceutical Sciences
Citation Excerpt :
In our model, genetic deficiency of Abcc2 had an about two times stronger influence on absorption and entero-systemic circulation of ezetimibe than chemical deficiency of Abcb1 as caused by PSC833. Associated to this apparent metabolic shift in transporter deficient animals, one has to consider that several cytochrome P450 enzymes were shown to be markedly higher expressed in Abcc2-deficient rats than wild-type animals, which may lead to several-fold increased formation of oxidative metabolites as shown for a tobacco-specific carcinogen by Leslie et al. 21–23 In well agreement to our conception, we found reduced sterol-lowering effects by inhibition of Abcb1, Abcc2 deficiency or the combination of both in parallel to decreased serum levels of ezetimibe.
Pharmacokinetics of the sterol-lowering drug ezetimibe (EZ) is influenced by intestinal ABCB1 and ABCC2. This study in Lew.1W rats with “chemical” and genetic Abcb1 and Abcc2 deficiency was initiated to evaluate the individual contribution of both efflux carriers to the overall disposition and sterol-lowering effects of EZ. Disposition and sterol-lowering effects of EZ (5 mg/kg, 14 days) were measured in wild-type (WT) and Abcc2-deficient (Abcc2-) rats (N = 8 per group) and in animals treated with PSC833 (20 mg/kg) to generate “chemical” Abcb1-deficiency (Abcb1-, Abcb1-/Abcc2-). EZ serum levels decreased in the order WT (3.11 ± 1.09 ng/mL), Abcb1- (1.94 ± 1.10 ng/mL), Abcc2- (1.42 ± 0.42 ng/mL, p = 0.003 vs. WT), Abcb1-/Abcc2- (1.17 ± 0.53 ng/mL, p = 0.002 vs. WT) whereas the serum EZ glucuronide levels increased as follows: WT (23.2 ± 24.6 ng/mL), Abcb1- (119 ± 74.5 ng/mL, p = 0.002 vs. WT), Abcc2- (195±76.5 ng/mL, p < 0.001 vs. WT), Abcb1-/Abcc2- (676 ± 207 ng/mL, p < 0.001 vs. WT, Abcb1- and Abcc2-). Abcb1 and Abcc2 protein deficiency resulted synergistically in lower fecal but increased renal excretion of total EZ although to a much lower extent. The sterol-lowering effects of EZ were significantly correlated to serum levels of EZ. In conclusion, Abcb1 and Abcc2 deficiency leads to lower levels of the active EZ and in turn to decreased sterol-lowering effects. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:422–429, 2010
Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes
2008, Toxicology in Vitro
Citation Excerpt :
APAP hepatotoxicity is also affected by changes in expression or activity of transporters involved in APAP metabolite disposition (Ghanem et al., 2005). Studies investigating APAP induced hepatotoxicity in transport deficient rat and mouse models underline the role of Mrp2 and Mrp3 in APAP resistance (Manautou et al., 2005; Silva et al., 2005). MRP2/Mrp2 and MRP3/Mrp3 are members of the multidrug resistance protein subfamily of transporters.
Most of the experiments studying acetaminophen (APAP) induced hepatotoxicity were performed using moue as model specie, right because its high sensitivity. While the toxic responses can be called forth easily in mice, the human relevancy of these results is questionable. In this study human, rat, and mouse primary hepatocytes were treated with increasing concentrations of APAP, and cell viability was measured by MTT cytotoxicity assay. Pronounced interspecies differences were obtained in cell viability following 24 h of APAP treatment starting at 24 h after seeding (EC₅₀: 3.8 mM, 7.6 mM, and 28.2 mM, in mouse, rat, and human hepatocyte culture, respectively). The longer time of culturing highly increased the resistance of hepatocytes of all species investigated. In rat hepatocyte culture EC₅₀ values were 6.0 mM, 12.5 mM, and 18.8 mM, when starting APAP treatment after 24, 48, and 72 h of seeding. Although N-acetylbenzoquinoneimine, a minor metabolite of APAP, which is mainly formed by CYP2E1 at high APAP concentration in every species studied, is thought to initiate the toxic processes, no correlation was found between CYP2E1 activities and hepatocyte sensitivity of different species. We conclude that the toxicity induced by APAP overdose highly depends on the animal model applied.
Modulation of trabectedin (ET-743) hepatobiliary disposition by multidrug resistance-associated proteins (Mrps) may prevent hepatotoxicity
2008, Toxicology and Applied Pharmacology
Trabectedin is a promising anticancer agent, but dose-limiting hepatotoxicity was observed during phase I/II clinical trials. Dexamethasone (DEX) has been shown to significantly reduce trabectedin-mediated hepatotoxicity. The current study was designed to assess the capability of sandwich-cultured primary rat hepatocytes (SCRH) to predict the hepato-protective effect of DEX against trabectedin-mediated cytotoxicity. The role of multidrug resistance-associated protein 2 (Mrp2; Abcc2) in trabectedin hepatic disposition also was examined. In SCRH from wild-type Wistar rats, cytotoxicity was observed after 24-h continuous exposure to trabectedin. SCRH pretreated with additional DEX (1 μM) exhibited a 2- to 3-fold decrease in toxicity at 100 nM and 1000 nM trabectedin. Unexpectedly, toxicity in SCRH from Mrp2-deficient (TR⁻) compared to wild-type Wistar rats was markedly reduced. Depletion of glutathione from SCRH using buthionine sulfoximine (BSO) mitigated trabectedin toxicity associated with 100 nM and 1000 nM trabectedin. Western blot analysis demonstrated increased levels of CYP3A1/2 and Mrp2 in SCRH pretreated with DEX; interestingly, Mrp4 expression was increased in SCRH after BSO exposure. Trabectedin biliary recovery in isolated perfused livers from TR⁻ rats was decreased by ∼ 75% compared to wild-type livers. In conclusion, SCRH represent a useful in vitro model to predict the hepatotoxicity of trabectedin observed in vivo. The protection by DEX against trabectedin-mediated cytotoxicity may be attributed, in part, to enhanced Mrp2 biliary excretion and increased metabolism by CYP3A1/2. Decreased trabectedin toxicity in SCRH from TR⁻ rats, and in SCRH pretreated with BSO, may be due to increased basolateral excretion of trabectedin by Mrp3 and/or Mrp4.
Role and Regulation of Hepatobiliary ATP-Binding Cassette Transporters during Chemical-Induced Liver Injury
2022, Drug Metabolism and Disposition

View all citing articles on Scopus

¹: Present address: Procter and Gamble Company, 6110 Center Hill Avenue 1B17, Cincinnati, OH 45249, USA.

²: Present address: Boehringer Ingelheim Pharmaceuticals Inc., Toxicology and Safety Assessment, 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877, USA.

View full text

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

Abstract

Introduction

Section snippets

Reagents

Animals

Studies on the susceptibility of TR− rats to APAP hepatotoxicity

Discussion

Acknowledgements

Toxicol Lett

J Biol Chem

Pharmacol Ther

Food Chem Toxicol

Hepatology

Toxicol Appl Pharmacol

Toxicology

Arch Biochem Biophys

Toxicol Appl Pharmacol

Toxicology

Toxicol Appl Pharmacol

Biochem Pharmacol

Pharmacol Ther

J Biol Chem

Life Sci

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

Drug Metab Dispos

Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(−) rats

J Pharmacol Exp Ther

Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene

Science

Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR

Am J Physiol

Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR−) with inherited conjugated hyperbilirubinemia

J Clin Invest

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

Studies on the susceptibility of TR⁻ rats to APAP hepatotoxicity

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

Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR⁻) with inherited conjugated hyperbilirubinemia