Shift from Biliary to Urinary Elimination of Acetaminophen- Glucuronide in Acetaminophen-Pretreated Rats Carolina I. Ghanem, Marı´a L. Ruiz, Silvina S. M. Villanueva, Marcelo G. Luquita, Viviana A. Catania, Brett Jones, Laura A. Bengochea, Mary Vore, and Aldo D. Mottino Ca ´tedra de Fisiopatologı´a, Departamento de Ciencias Biolo ´gicas, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Buenos Aires, Argentina (C.I.G., L.A.B.); Instituto de Fisiologı´a Experimental, Facultad de Ciencias Bioquı´micas y Farmace ´ uticas, Consejo Nacional de Investigaciones Cientificas y Tecnicas-Universidad Nacional de Rosario, Rosario, Argentina (M.L.R., S.S.M.V., M.G.L., V.A.C., A.D.M.); and Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky (B.J., M.V.) Received June 6, 2005; accepted August 15, 2005 ABSTRACT Despite its toxicity, acetaminophen (APAP) is used increasingly as an analgesic, antipyretic, and anti-inflammatory agent. We examined the effect of prior exposure to APAP on its biliary and urinary elimination. The biliary and urinary elimination of a test dose of APAP (150 mg/kg i.v.) was determined in male Wistar rats 24 h after pretreatment with vehicle, a single dose (1.0 g/kg i.p.), or increasing daily doses (0.2, 0.3, 0.6, and 1.0 g/kg/day i.p.) of APAP. Although elimination of the parent APAP was minimally affected, biliary excretion of APAP glucuronide was significantly decreased 70 and 80%, whereas urinary excretion was significantly increased 90 and 100% in the groups pre- treated with single and repeated doses of APAP, respectively, relative to vehicle controls. Western analysis and confocal im- munofluorescent microscopy indicated a marked increase in hepatic expression of multidrug resistance-associated protein 3 (Mrp3) in both groups pretreated with APAP, relative to ex- pression of Mrp2. ATP-dependent transport of [ 3 H]tauro- cholate, an Mrp3 substrate, was significantly increased in ba- solateral liver plasma membrane vesicles from rats pretreated with repeated doses of APAP relative to controls. Enterohepatic recirculation of APAP glucuronide after administration of the same test dose of the drug was significantly decreased in rats pretreated with repeated doses of APAP. These data indicate that APAP pretreatment induced a shift from biliary to urinary elimination of APAP glucuronide, consistent with the increased expression of Mrp3 in the basolateral domain of the hepato- cyte. We postulate that decreased enterohepatic recirculation contributes to decreased APAP hepatotoxicity by reducing liver exposure. The increasing popularity of acetaminophen (N-acetyl-p- aminophenol, APAP) as a nonprescription analgesic and an- tipyretic has resulted in a significant increment in the inci- dence of accidental and intentional poisonings with this drug (Kaplowitz, 2004). It is generally accepted as a safe drug when administered within the therapeutic range (Rumack, 2004). However, in large single-dose ingestion, it causes mas- sive centrilobular necrosis, inducing severe hepatotoxicity in human and experimental animals that may be lethal (Thomas, 1993; Bessems and Vermeulen, 2001). APAP is metabolized in the liver mainly by glucuronida- tion and sulfation, thus generating the nontoxic metabolites, APAP-glucuronide (APAP-glu) and APAP sulfate (Thomas, 1993). At high doses of APAP, these metabolic paths are saturated and a substantial portion of the drug is metabo- lized by the CYP450 system, generating a reactive metabo- lite, NAPQI. This compound reacts with reduced GSH, lead- ing to GSH depletion, or it covalently binds to cellular proteins, as likely explanations for APAP hepatotoxicity (Mitchell et al., 1973; Linscheer et al., 1980). APAP-glu ex- cretion in bile is mediated by the canalicular multidrug re- sistance-associated protein 2 (Mrp2; Abcc2) (Xiong et al., 2000) and represents approximately 7% of a single dose (100 mg/kg) of APAP administered systemically to rats (Brouwer and Jones, 1990). Basolateral efflux of APAP-glu in liver may also occur and has been linked to the expression of Mrp3 This work was supported by Grants from Agencia Nacional de Promocio ´n Cientı ´fica y Tecnolo ´gica, Consejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas (to A.D.M.), by Grants B036 and B045 from Universidad de Buenos Aires (to L.A.B.), and by Public Health Service Grant GM55343 (to M.V.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.090613. ABBREVIATIONS: APAP, acetaminophen; APAP-glu, acetaminophen-glucuronide; Mrp1, Mrp2, and Mrp3, multidrug resistance-associated proteins 1, 2, and 3; NAPQI, N-acetyl-p-benzoquinone imine; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GST, glutathione S-transferase; GSH, glutathione; GSSG, oxidized glutathione; TC, taurocholate; UGT, UDP-glucuronosyltransferase. 0022-3565/05/3153-987–995$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 315, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 90613/3060368 JPET 315:987–995, 2005 Printed in U.S.A. 987 by guest on November 27, 2013 jpet.aspetjournals.org Downloaded from