THE ROLE OF PREGNANE X RECEPTOR IN 2-ACETYLAMINOFLUORENE-MEDIATED INDUCTION OF DRUG TRANSPORT AND -METABOLIZING ENZYMES IN MICE Alexander Anapolsky, Shirley Teng, Santosh Dixit, and Micheline Piquette-Miller Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada (A.A., S.T., M.P.-M.); and Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee (S.D.) Received June 22, 2005; accepted December 14, 2005 ABSTRACT: Activation of the pregnane X receptor (PXR) mediates the induction of several drug transporters and -metabolizing enzymes. In vitro studies have reported that several of these genes are induced after exposure to the hepatocarcinogen, 2-acetylaminofluorene (2-AAF). Thus, we hypothesized that PXR may play a role in the in vivo induction of gene expression by 2-AAF. We examined the expres- sion of the drug-metabolizing enzymes CYP1A2 and CYP3A11 and the drug transporters breast cancer resistance protein (BCRP), MRP2, and OATP2. Wild-type (PXR / ) and PXR-null (PXR / ) C57BL/6 mice were injected daily for 7 days with 150 or 300 mg/kg 2-AAF suspended in corn oil (i.p.), whereas the control group re- ceived corn oil vehicle. Levels of mRNA isolated from liver were measured by reverse transcription-polymerase chain reaction and normalized to -actin. Treatment of PXR / mice resulted in a dose-dependent 2- to 4-fold induction (p < 0.001) of MRP2, OATP2, BCRP, CYP3A11, and CYP1A2, but no induction was observed in PXR / mice. Induction of PXR mRNA was observed in the 2-AAF- treated PXR / mice. Furthermore, a dose-dependent increase in CYP3A4 promoter construct activity was observed in HepG2 cells cotransfected with human or rat PXR, indicating that 2-AAF does indeed activate PXR. These results suggest that PXR is responsi- ble for 2-AAF-mediated induction of drug efflux transporters and biotransformation enzymes in the liver. Moreover, novel findings demonstrate that PXR plays a role in regulation of the drug efflux transporter, BCRP, in mice. Short-term administration of the polycyclic aromatic amine, 2-acetylaminofluorene (2-AAF), in rodents is associated with pre- neoplastic changes in hepatocytes and the development of a drug- resistance phenotype (Gant et al., 1991). The hydroxylated metab- olite of 2-AAF, produced predominantly by CYP1A2, has been shown to account for genotoxicity (Russell et al., 1994; Schrenk et al., 1994). Several studies have suggested that exposure to 2-AAF causes induction of the multidrug-resistant transporters MRP2 (Kauffmann et al., 1997) and MDR1 (Teeter et al., 1993; Schrenk et al., 1994), as well as the drug-metabolizing enzyme CYP3A23 (Sparfel et al., 2003). The overlap in genes induced by 2-AAF suggests a common molecular mechanism responsible for regulation of their expression. In particular, it is believed that the pregnane X receptor (PXR) may be responsible for this induction (Kliewer et al., 1998). Genes shown to be regulated by PXR include the ABC drug transporters MDR1 (Geick et al., 2001), MRP2 (Kast et al., 2002), and MRP3 (Teng et al., 2003), the organic anion transporter OATP2 (Staudinger et al., 2001), as well as the CYP3A drug-metabolizing enzyme. Recent in vitro studies have shown that administration of 2-AAF elicits a PXR- dependent induction of MRP2 and CYP3A23 (Kauffmann and Schrenk, 1998; Sparfel et al., 2003). In vivo studies elucidating the effects of 2-AAF on murine gene up-regulation have yet to be com- pleted. Therefore, we examined the in vivo effects produced by 2-AAF administration on the expression of several murine hepatic genes which encode for active drug transporters and drug metaboliz- ing enzymes. Novel findings from this study revealed a 2-AAF- mediated dose-dependent induction of the organic anion drug trans- porters MRP2 and OATP2, and the CYP3A11 and CYP1A2 drug metabolizing enzymes in wild-type (PXR +/+ ), but not in PXR-null (PXR -/- ) mice, demonstrating involvement of murine PXR (PXR) in the regulatory effects of 2-AAF. Activation of PXR by 2-AAF was further substantiated by CYP3A4 promoter construct studies which demonstrated an increase in luciferase reporter gene activity in HepG2 cells cotransfected with human or rat PXR. Moreover, the observed 2-AAF-mediated induction of the breast cancer resistance protein (BCRP) in PXR +/+ , but not in PXR -/- mice, is the first finding to demonstrate involvement of PXR in the regulation of this novel ABC-half-transporter. Materials and Methods In Vivo Studies. All animal studies were approved by the University of Toronto Animal Ethics Committee and were conducted in accordance with the guidelines of the Canadian Council on Animal Care. Wild-type (PXR +/+ ), 8-week-old C57BL/6 mice (25–30 g) were purchased from Charles River Canada (St. Constant, QC, Canada); PXR-null (PXR -/- ) mice colonies were Funding for this study was provided by a grant from the Canadian Institutes of Health Research (CIHR). Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.006197. ABBREVIATIONS: 2-AAF, 2-acetylaminofluorene; PXR, pregnane X receptor; MRP, multidrug resistance-associated protein; PCR, polymerase chain reaction; OATP, organic anion transporting peptide; BCRP, breast cancer resistance protein; RU486, 17-hydroxy-11-[4-dimethylamino phenyl]-17-[1-propynyl]estra-4,9-dien-3-one; P450, cytochrome P450; ABC, ATP-binding cassette transporter; AhR, aryl hydrocarbon receptor. 0090-9556/06/3403-405–409$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 3 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 6197/3090562 DMD 34:405–409, 2006 Printed in U.S.A. 405 at ASPET Journals on August 17, 2017 dmd.aspetjournals.org Downloaded from