Transport deficient (TR−) hyperbilirubinemic rats are resistant to acetaminophen hepatotoxicity
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.
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Present address: Procter and Gamble Company, 6110 Center Hill Avenue 1B17, Cincinnati, OH 45249, USA.
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Present address: Boehringer Ingelheim Pharmaceuticals Inc., Toxicology and Safety Assessment, 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877, USA.