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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)
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Abstract |
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). The hydroxylated metabolite 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 completed. 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 metabolizing enzymes. Novel findings from this study revealed a 2-AAF-mediated dose-dependent induction of the organic anion drug transporters 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.
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Materials and Methods |
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Two PXR+/+ and two PXR–/– groups of mice (n = 4 per group) were treated intraperitoneally (i.p.) for 7 days with 150 mg/kg or 300 mg/kg 2-AAF (Sigma-Aldrich, Oakville, ON, Canada) suspended in corn oil. Each control group (n = 4–8 per group) was treated i.p. with corn oil vehicle using the same dosing schedule. On day 8, all animals were sacrificed by cervical dislocation, and their livers were removed, snap-frozen in liquid nitrogen, and stored at –80°C until used for RNA isolation. Normal serum alanine aminotransferase levels were found in all animals treated with these doses of 2-AAF, indicating the absence of liver necrosis.
Total RNA was extracted from control and 2-AAF-treated liver using the GE Healthcare Quick-Prep RNA isolation kit (GE Healthcare, Little Chalfont, UK) according to the manufacturer's protocol. cDNA was synthesized from total RNA (0.5 µg) using the First Strand cDNA Synthesis Kit (MBI Fermentas, Flamborough, ON, Canada), according to the manufacturer's protocol. PCR standard curves for each gene product (ß-actin, BCRP, CYP3A11, CYP1A2, MDR1a, MDR1b, MRP2, OATP2, and PXR) were generated as previously reported (Teng and Piquette-Miller, 2004). PCR was performed in the presence of 3 mM MgCl2, 200 µM deoxynucleoside-5'-triphosphate, and 50 pmol of each primer in a total volume of 50 µl using a GeneAmp 2400 Thermocycler (PerkinElmer, Mississauga, ON, Canada). The reactions were initiated by the addition of 1.5 units of Taq polymerase (MBI Fermentas), and amplification proceeded through 24 cycles for BCRP, 28 cycles for CYP3A11, 22 cycles for CYP1A2, 33 cycles for MDR1a and MDR1b, 26 cycles for MRP2, 19 cycles for OATP2, and 28 cycles for PXR. PCR products were run on a 2% agarose gel, stained with SYBR Gold (Invitrogen, Carlsbad, CA), and quantitated using Kodak Digital Science1D Image Analysis software. Sizes of DNA bands were confirmed using the Gene Ruler 100-bp DNA ladder (MBI Fermentas). All mRNA levels were normalized to ß-actin mRNA. Levels of MRP2, OATP2, BCRP, CYP3A11, CYP1A2, and PXR mRNA expression are reported as percentages of normalized values, as compared with controls. Data are presented as a mean value with standard error of the mean (S.E.M.). Differences between PXR+/+, PXR–/– treatment groups, and controls were determined by one-way analysis of variance, followed by Tukey's test with a significance level of *, p < 0.05 or **, p < 0.001, using SPSS Statistical Software (Version 11.0.0, SPSS Inc., Chicago, IL).
Luciferase Reporter Assay. A CYP3A4-XREM-Luc reporter plasmid driven by CYP3A4 regulatory elements (–7836/–7208) (Goodwin et al., 1999) in pGL3 Basic vector (Promega, Madison, WI) was prepared as described previously (Tirona et al., 2003). Human PXR was cloned in pEF6/V5-His expression vector (Invitrogen) as described previously (Zhang et al., 2001). A rat PXR expression plasmid was obtained by PCR from a rat liver cDNA library (BD Biosciences Clontech, Palo Alto, CA) and subsequent cloning into pEF6/V5-His, as previously described (Tirona et al., 2004).
Human hepatocarcinoma HepG2 cells (American Type Culture Collection, Manassas, VA) were grown in 75-cm2 tissue culture flasks in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine system (Sigma) and 50 U/ml penicillin-streptomycin (Invitrogen). Cells were cultured overnight at 37°C and then trypsinized and reseeded at 3 x 105 cells/well in a 24-well plate (Corning Inc., Corning, NY) in Dulbecco's modified Eagle's medium containing 10% fetal bovine system. After 24 h, cells were transfected using Lipofectin (Invitrogen) with 250 ng/well of CYP3A4-XREM-Luc, 250 ng/well of the human PXR or rat PXR expression plasmids, or pEF6 vector lacking cDNA insert, respectively. All wells were also cotransfected with a Renilla luciferase vector driven by a cytomegalovirus promoter (7.5 ng/well of pRL-CMV) as an internal control for transfection efficiency. Sixteen hours thereafter, cells were treated with 2-AAF (1 µM-100 µM) or vehicle (dimethyl sulfoxide, 0.1%) for 48 h. Luciferase activity was determined with the Dual Luciferase Assay Kit (Promega) according to the manufacturer's instructions. Statistical differences between triplicate control and 2-AAF-treated cells were determined using Student's t test, with a p value of <0.05 taken to be the minimum level of statistical significance.
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Results |
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As shown in Fig. 2, levels of PXR mRNA were increased in 2-AAF-treated PXR+/+ mice with a significant 1.8-fold induction of PXR mRNA observed in the 300 mg/kg 2-AAF PXR+/+ mice (p < 0.05). Compared with PXR +/+ mice, basal levels of MRP2 and BCRP were significantly lower (30–33% of wild types) and levels of OATP2, significantly higher (2 fold), in PXR–/– mice. Basal levels of CYP1A2 and CYP3A11 were not significantly different between PXR+/+ and PXR–/– vehicle-treated mice. No relationship was observed between basal mRNA expression to gene induction in the 2-AAF-treated PXR+/+ and PXR–/– mice, which is in agreement with that seen in PCN-treated mice (Teng and Piquette-Miller, 2004).
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Discussion |
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; Lehman et al., 1998). These findings were the first to suggest that common signal transduction pathways could be involved in the molecular regulation of both drug transport and -metabolizing enzymes. Characterization of the nuclear receptor PXR in 1998 began to delineate the underlying regulatory molecular mechanisms involved (Kliewer et al., 1998). Since that time, various studies have shown that induction of CYP3A by a variety of xenobiotics is primarily mediated by PXR (Bertilsson et al., 1998; Lehman et al., 1998; Guo et al., 2003). Studies performed with murine PXR showed that it is efficiently activated by the classic CYP3A inducer pregnenolone 16
-carbonitrile, as well as by glucocorticoids such as dexamethasone and spironolactone (Drocourt et al., 2001), the antiglucocorticoid RU486 (Staudinger et al., 2001; Xie et al., 2001), the HIV-1 protease inhibitor ritonavir (Dussault et al., 2001), and the anticancer drug paclitaxel (Synold et al., 2001). The "promiscuity" of PXR toward ligands might contribute to the ability of various classes of xenobiotics to coinduce both cytochrome P450 (P450) and drug efflux transporters (Goodwin et al., 2002).
Located on the canalicular membrane of hepatocytes, MRP2 is primarily responsible for biliary elimination of non-bile acids and organic anions into bile. Results from this study demonstrated a dose-dependent induction of MRP2 in 2-AAF-treated PXR+/+, but not in PXR–/– mice. This implies both that 2-AAF may be a potent activator of PXR in vivo and that induction of MRP2 via 2-AAF likely occurs through a PXR-mediated pathway. Based on the ability of rifampicin to induce both murine and human CYP3A (Kliewer et al., 1998) and MRP2 (Fromm et al., 2000), it has been hypothesized that PXR could be the putative regulator of their transcription. Other investigators have shown a concentration-dependent up-regulation of MRP2 by 2-AAF in primary cultured rat hepatocytes and human HepG2 cells (Kauffmann et al., 1997; Schrenk et al., 2001). More importantly, studies conducted in knockout mice have demonstrated induction of MRP2 in PXR+/+ but not PXR–/– animals after the administration of various other PXR ligands (Kast et al., 2002; Teng and Piquette-Miller, 2004), demonstrating involvement of PXR in MRP2 induction.
Our results demonstrate a 2-AAF-mediated induction of OATP2 mRNA in PXR+/+ mice, but not in PXR–/– mice, providing another line of evidence that 2-AAF exerts its effects on gene expression through PXR in vivo. OATP2, a basolateral transporter mediating hepatocellular uptake of bile acids and a wide range of xenobiotics, was initially isolated from rat brain (Noe et al., 1997) and, later, was found to be abundantly expressed in rodent liver (Reichel et al., 1999). In addition to MRP2 and CYP3A11, OATP2 has been implicated in bile acid synthesis, transport, and metabolism (Noe et al., 1997; Reichel et al., 1999). Hence, it has been hypothesized that PXR might be directly involved in regulation of OATP2 gene transcription. Our findings are supported by earlier studies, which have reported a coinduction of CYP3A11 and OATP2 in PXR+/+ mice, but not in the PXR–/– model, after treatment with the known PXR activators RU486 and pregnenolone 16-carbonitrile (Staudinger et al., 2001; Teng and Piquette-Miller, 2004).
Interestingly, the observed induction of the breast cancer resistance protein, BCRP, in 2-AAF-treated PXR+/+ mice, which was not seen in 2-AAF-treated PXR–/– mice, suggests that a PXR-dependent molecular mechanism may be involved in regulating BCRP expression. Transcriptional regulation of BCRP has not been previously established. BCRP, which is one of the most recently discovered ABC-drug efflux transporters, was first cloned from a doxorubicin-resistant MCF7 breast cancer cell line. BCRP is normally found on apical membranes of intestinal, hepatic, and brain epithelia involved in drug disposition. Overexpression of BCRP, seen in various cancer cells and stem cells, has been shown to cause multidrug resistance. Mouse and human cell lines expressed with BCRP have been demonstrated to extrude anticancer agents that overlap considerably with substrates for MRP2 (Schinkel and Jonker, 2003). Furthermore, BCRP has been shown to share structural similarity with MRP2 and other ABC transporters, but, unlike them, it is a half-transporter and probably mediates its drug efflux functions either by homo- or heterodimerization (Kage et al., 2002; Schinkel and Jonker, 2003). Recent investigations reported the existence of a putative estrogen response element in the promoter region of BCRP in estrogen receptor-positive cells (Ee et al., 2004). Expression of BCRP has also been shown to be up-regulated by hypoxia-inducible transcription factor complex HIF-1, suggesting the cytoprotective role of BCRP during hypoxia (Krishnamurthy et al., 2004). Recent studies indicate that BCRP, in addition to MRP1 and MRP3, may be relevant to hepatic cell survival during carcinogenesis (Zhou et al., 2002; Ros et al., 2003). Thus, in addition to structural resemblance, a dose-dependent induction of both BCRP and MRP2 in the present study suggests a similarity in function of these transporters during the administration of 2-AAF.
The cytochrome P450 enzymes (P450s) are a superfamily of hemethiolate proteins that play a central role in the oxidative, peroxidative, and reductive metabolism of a large spectrum of endogenous compounds such as fatty acids, steroids, leukotrienes, prostaglandins, bile acids, and fat-soluble vitamins. In addition, many of these enzymes are responsible for the detoxification of xenobiotics such as drugs, carcinogens, and environmental contaminants (Bertilsson et al., 1998; Kliewer et al., 1998). Because we observed induction of CYP3A11 and CYP1A2 in the 2-AAF-treated PXR+/+, but not PXR–/– mice, our results indicate that PXR is involved in the in vivo induction of CYP3A11 and CYP1A2 imposed by 2-AAF. This supports previous in vitro findings of a 2-AAF-mediated up-regulation of CYP1A and CYP3A2/23 in primary rat hepatocytes (Tateishi et al., 1999; Sparfel et al., 2003). It has been generally accepted that polycyclic aromatic molecules such as 2-AAF are agonists of the aryl hydrocarbon receptor (AhR). Activation of the AhR has been linked to alterations in CYP1A1 and CYP1A2 mRNA levels during carcinogenesis (Cikryt et al., 1990; Gant et al., 1991). C57BL/6 mice, used in the present study, possess AhRs with a high affinity for aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholantrene. Initial in vivo studies have shown that both chemicals induce CYP1A in mice (Gonzalez et al., 1984). Although affinity of 2-AAF for the AhR and an up-regulation of CYP1A have been shown in rats (Cikryt et al., 1990; Tateishi et al., 1999), the mechanistic role of AhRs in 2-AAF-induced CYP1A2 induction has not been established (Gant et al., 1991; Tateishi et al., 1999). Our results demonstrating an expected increased CYP1A2 expression in 2-AAF-treated PXR+/+ mice, but a complete lack of CYP1A2 induction in 2-AAF-treated PXR–/– mice, indicates that PXR may play a more important role in CYP1A2 regulation than putative AhR in vivo. Induction of the multidrug resistance gene, MDR1, along with MRP2 and CYP1A1, has been reported during 2-AAF-induced carcinogenesis (Burt and Thorgeirsson, 1988; Gant et al., 1991; Kauffmann et al., 1997; Tateishi et al., 1999). Although we did not detect significant changes in mRNA levels of MDR1a or MDR1b in the present study, numerous species- and strain-specific differences in 2-AAF-mediated induction of MDR1 have been reported (Lecureur et al., 1996). These differences are felt to stem primarily from the finding that the CYP1A2 metabolites of 2-AAF, N-hydroxylated 2-AAF, and N-acetoxy-2-acetylaminofluorene, are responsible for MDR1 induction (Schrenk et al., 1994; Hill et al., 1996). Whether the 2-AAF-mediated induction of the drug transporters or drug-metabolizing enzymes observed in this study occurs because of 2-AAF or its metabolites remains to be determined.
To confirm whether observed PXR-dependent changes in transporter and P450 expression occurred as a result of direct activation of PXR by 2-AAF, in vitro reporter gene assays were performed in HepG2 cells cotransfected with a PXR-responsive CYP3A4-luciferase reporter and either the rat PXR or human PXR expression plasmids. Indeed, 2-AAF was found to be a highly efficacious activator of rat PXR. Interestingly, 2-AAF was also able to activate human PXR, but at higher concentrations. Of note, many compounds are able to activate PXR in different species; in particular, ligand specificity is almost entirely shared between rats and mice. Thus, although these in vitro studies examined the activation of rat and human PXR, whereas our in vivo studies were performed in mice, it is very likely that 2-AAF also serves as a PXR ligand in mice. Taken together, these findings suggest that 2-AAF is a ligand of PXR, and the observed induction of drug transporters and P450s upon exposure to this compound is likely mediated through activation of PXR.
In conclusion, our findings demonstrated a dose-dependent induction in the hepatic expression of the drug transporters MRP2, OATP2, and BCRP and the CYP3A11 and CYP1A2 drug-metabolizing enzymes in 2-AAF-treated PXR+/+, but not PXR-null mice. Cell-based reporter assays confirmed that 2-AAF serves as a ligand of PXR. Thus, induction of these genes occurs as a result of the activation of PXR by 2-AAF. Further studies elucidating the impact of 2-AAF on the activity of these genes and its impact on the drug disposition are warranted.
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Acknowledgments |
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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, 17ß-hydroxy-11ß-[4-dimethylamino phenyl]-17-[1-propynyl]estra-4,9-dien-3-one; P450, cytochrome P450; ABC, ATP-binding cassette transporter; AhR, aryl hydrocarbon receptor.
Address correspondence to: Dr. M. Piquette-Miller, Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, Canada, M5S 2S2. E-mail: m.piquette.miller{at}utoronto.ca
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