ToxSci Advance Access originally published online on March 17, 2006
Toxicological Sciences 2006 91(2):448-455; doi:10.1093/toxsci/kfj163
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The Mycoestrogen Zearalenone Induces CYP3A through Activation of the Pregnane X Receptor
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, Kansas 66045
1 To whom correspondence should be addressed at the department of Pharmacology and Toxicology, University of Kansas, 5044 Malott Hall, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045. Fax: (785) 864-5219. E-mail: stauding{at}ku.edu.
Received January 31, 2006; accepted March 2, 2006
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ABSTRACT |
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Zearalenone is a mycoestrogen that is produced in the fungi Fusarium graminearum, Fusarium culmorum, Fusarium equiseti, and Fusarium crookwellense. These fungi commonly exist in agricultural products. Human pregnane X receptor (hPXR) is a ligand-activated transcription factor that regulates the expression of numerous hepatic drug-metabolizing enzymes, including several clinically important cytochrome P450s. In this report, we show that zearalenone is an efficacious ligand for hPXR. We also describe the creation and validation of a novel adenoviral-mediated transduction protocol used to express functional FLAG-tagged-hPXR protein in a transformed cell line (HepG2) and primary cell types (cultured hepatocytes). Treatment of hPXR-transduced HepG2 cells with zearalenone induces expression of CYP3A4, the "prototypical" PXR-target gene in human liver. Treatment of hPXR-transduced cultured hepatocytes isolated from PXR-knockout mice with zearalenone induces the expression of Cyp3a11, the prototypical murine hepatic PXR-target gene. Using mammalian two-hybrid assays, we show that zearalenone displaces the nuclear receptor corepressor protein N-CoR from hPXR, while it recruits coactivator proteins steroid receptor coactivator-1, Glucocorticoid Receptor-Interacting Protein 1 and PPAR-Binding protein (GRIP1) and PBP to hPXR. Concentration-response analysis using a PXR-responsive reporter gene assay reveals that zearalenone activates hPXR with an EC50 value of approximately 1.5 µM. Because activation of hPXR represents the molecular basis of an important class of drug interactions, our findings suggest that studies to investigate the potential of zearalenone to induce the metabolism of other drugs in humans are warranted. In addition, due to the limited availability of primary human hepatocytes, our adenoviral-mediated hPXR expression protocol will likely prove useful in studies of the xenobiotic response.
Key Words: PXR; CYP3A; zearalenone; drug interaction; cofactor; nuclear receptor.
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INTRODUCTION |
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Zearalenone (6-[10-hydroxy-6-oxo-trans-1-undecenyl]-B-resorcyclic acid lactone) is a mycoestrogen that activates the estrogen receptor (ER) with an efficacy comparable to that of 17ß-estradiol, the principle endogenous ER ligand. Zearalenone is biosynthesized by the fungi Fusarium graminearum, Fusarium culmorum, Fusarium equiseti, and Fusarium crookwellense (Bennett and Klich, 2003
). These fungi are commonly found in cereal crops worldwide. Zearalenone has also been patented as an oral contraceptive (Bennett and Klich, 2003). While most of its biological activities are attributed to the agonist effect on the ER, zearalenone also produces certain biological reactions that cannot be explained by its estrogenic activity (Hidy et al., 1977; Utian, 1973). To further explore the biological activities of zearalenone, we screened a panel of nuclear receptor family members for their ability to respond to this compound. We found that in addition to ER
, zearalenone also efficaciously activates the human xenobiotic receptor—pregnane X receptor (PXR, NR1I2).
PXR is a member of the nuclear receptor superfamily of ligand-activated transcription factors (Blumberg et al., 1998; Kliewer et al., 1998). It is a key regulator of xenobiotic-inducible CYP3A gene expression (Goodwin et al., 2002; Kliewer et al., 2002). In addition, it regulates other genes involved in the metabolism of xenobiotic and endobiotic compounds such as CYP2B, CYP2C, CYP24, glutathione S-transferases, sulfotransferases, and Uridine diphosphate (UDP)-glucuronosyltransferases (Chen et al., 2003; Maglich et al., 2002; Pascussi et al., 2005; Sonoda et al., 2002; Wei et al., 2002). PXR also regulates the expression of the drug transporter genes Oatp2, Mdr1, Mrp2, and Mrp3 (Geick et al., 2001; Kast et al., 2002; Staudinger et al., 2003). Therefore, PXR activation has a dual nature. On one hand, it protects cells from toxic insults. On the other, it represents the molecular basis for an important class of drug interactions.
In the present report, we show that zearalenone activates human PXR (hPXR) and induces CYP3A4 in HepG2 cells in a PXR-dependent manner. The PXR-dependent induction of CYP3A was further confirmed by utilizing primary mouse hepatocytes isolated from PXR-KO mice and transduced with adenovirus carrying either Green Flourescent Protein (GFP) (blank virus) or hPXR. Moreover, we demonstrate that at a molecular level, zearalenone activates PXR by displacing the corepressor N-CoR and by recruiting the coactivator proteins steroid receptor coactivator-1 (SRC-1), PBP, and GRIP1. Our data suggest that the exposure to zearalenone likely increases the metabolism of coadministered drugs and potentially causes food-drug interaction.
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MATERIALS AND METHODS |
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Animal care.
Generation of the PXR-knockout (PXR-KO) mice was previously described (Staudinger et al., 2001
). All rodents were maintained on standard laboratory chow and were allowed food and water ad libitum. The studies reported here have been carried out in accordance with the Declaration of Helsinki and/or with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.
Plasmids and chemicals.
The full-length hPXR, mouse PXR, mouse PXR2, human CAR, mouse CAR, and human RXR mammalian expression vectors were described previously (Kliewer et al., 1998; Lehmann et al., 1997, 1998). Gal4-fused human ER-LBD, GR-LBD, and mouse PPAR
-LBD mammalian expression vectors were described previously (Goodwin et al., 2000; Kliewer et al., 1997; Oliver et al., 2001). XREM-Luc and RXRE-Luc were described previously (Goodwin et al., 1999; Kliewer et al., 1992). pFR-Luc is commercially available (Stratagene, La Jolla, CA). VP16-hPXR mammalian expression vector was described previously (Ding and Staudinger, 2005a). GAL4-SRC-1, GAL4-PBP, and GAL4-N-CoR were generous gifts from Dr. Barry Forman (Synold et al., 2001). GRIP1 RID was polymerase chain reaction (PCR) amplified from pGAD-424-GRIP1 (Hong et al., 1997) (a generous gift from Dr. Stallcup) using the following primers: left primer, 5' gAC ggC GAA TTC ATg CCC CAg gCg gCC AgC ggg 3', and right primer, 5' gAC ggC ggA TCC TCA gAg TTT ggg ggT TAT TTC Cgg 3'. To generate GAL4-GRIP1, PCR-amplified GRIP1 RID was cloned into the EcoRI and BamHI sites of pM (BD Biosciences, Palo Alto, CA). The final construct was verified by DNA sequencing. The SV-ß-Gal plasmid is commercially available (Invitrogen, Carlsbad, CA). All compounds were purchased from Sigma (St. Louis, MO) and were dissolved as 1000X stocks in DMSO.
Cell culture and transient transfection of CV-1 cells.
CV-1 cells were plated on 96-well plates as described previously (Brobst et al., 2004). The XREM-LUC reporter gene assays and mammalian two-hybrid assays were performed as described previously (Brobst et al., 2004; Ding and Staudinger, 2005a,c). For RXRE-LUC and pFR-LUC assays, each well was transfected with 20 ng of reporter gene, 5 ng of nuclear receptor expression vector (human RXR for RXRE-LUC, GAL4-human ER-LBD, GAL4-human GR-LBD, and GAL4-mouse PPAR-LBD for pFR-LUC), and 40 ng of SV-ß-gal and added with pBluescript to 110 ng of total DNA per well. Twenty-four hours posttransfection, cells were drug treated for 24 h. The luciferase activities were determined using the luciferase assay kit per the manufacturer's instructions (Promega, Madison, WI) and normalized to ß-galactosidase acivities. ß-Galactosidase acivities were measured using o-nitrophenyl ß-D-galactopyranoside (ONPG) assay (Sigma). For ONPG assay, 110 mg ONPG was dissolved in 100 ml 0.1 M NaHPO4 buffer, which was made by mixing 6.84 ml 1 M Na2HPO4, 3.16 ml 1 M NaH2PO4, and 90 ml H2O. Twenty microliters of cell lysate and 200 µl ONPG buffer were mixed and incubated at 37°C for 30–60 min and read at 420 nm.
Real-time quantitative PCR.
Mouse hepatocytes were isolated using a two-step perfusion as described previously (Ding and Staudinger, 2005b). About 48 h postplating, the hepatocytes were transduced with adenovirus. Twenty-four hours later, hepatocytes were treated with drugs in maintenance medium for additional 24 h before RNA isolation. HepG2 cells were plated on a 12-well plate at a density of 5 x 105 cells per well in Dulbecco's Modified Eagle's Medium (DMEM) (Mediatech, Herndon, VA) supplemented with penicillin-streptomycin (100 units/ml penicillin and 100 µg/ml streptomycin) and L-glutamine (2 mM). About 24 h postplating, the hepatocytes were transduced with adenovirus. Twenty-four hours later, HepG2 cells were treated with drugs in maintenance medium for additional 24 h before RNA isolation. Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA (10 µg/lane) was resolved on 3.7% formaldehyde and 1% agarose gel to verify the integrity of the RNA. One microgram of DNaseI-treated RNA was reverse transcribed using random primers following the manufacturer's instructions (Promega). Equal amounts of cDNA were used in real-time quantitative PCRs. Reactions included 200 nM fluorogenic probe and 300 nM primers specific for each gene. The fluorogenic probe and primer sets were designed using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). BioSearch Technologies (Novato, CA) synthesized the fluorogenic probes. The sequences (5' to 3') for the primers and probes are as follows: CYP3A4, forward primer (CAg gAg gAA ATT gAT gCA gTT TT), fluorogenic probe (FAM-CCC AAT AAg gCA CCA CCC ACC TAT gA-BHQ1), and reverse primer (gTC AAg ATA CTC CAT CTg TAg CAC AgT); Cyp3a11, forward primer (CAA ggA gAT gTT CCC TgT CA), fluorogenic probe (FAM-AgA Agg CAA AgA AAg gCA AgC CTg-BHQ1), and reverse primer (CCA CgT TCA CTC CAA ATg AT). For 18S, 1X SybrGreen (BioWhittaker Molecular Applications, Rockland, ME) was included in the reaction instead of fluorogenic probe. The sequences (5' to 3') for the 18S primers are as follows: forward primer (AgT CCC TgC CCT TTg TAC ACA) and reverse primer (CgA TCC gAg ggC CTC ACT A). Cycling conditions were 95°C for 2 min followed by 45 cycles of 95°C for 15 s, 60°C for 15 s, and 68°C for 15 s using the Cepheid Smart Cycler system (Sunnyvale, CA). Fold induction was calculated as described (Schmittgen et al., 2000).
Adenovirus transduction and Western blot assay.
Ad-GFP (Lehman et al., 2000) was a generous gift from Dr. Daniel Kelly (Washington University). To generate Ad-hPXR, full-length hPXR was PCR amplified from pSG5-hPXR. PCR product was then cloned into the BglII and XhoI sites of pShuttle-IRES-hrGFP (Stratagene), which produced an in-frame fusion of PXR and the FLAG tag in the vector. The final construct was verified by DNA sequencing. Recombination was performed using the AdEasy system according to the manufacturer's instruction (Stratagene). Adenovirus was amplified in HEK293 cells as described (He et al., 1998). After amplification, virus was purified using two-step CsCl gradient centrifugation. Since the cesium chloride has a toxic effect on cell cultures, the virus suspension was dialyzed extensively against four changes of buffer (10 mM Tris-HCL pH 8.0, 100 mM NaCl, 0.1% bovine serum albumin, 20% glycerol), for 4 h each of dialysis. Cells were transduced for 48 h before protein isolation. Whole cell lysate was prepared in the lysis buffer containing 10% glycerol, 1% NP-40, 20 mM Tris (pH = 8), 137 mM NaCl, 1 mM NaVn, and 1X Complete (Roche Diagnostics, Indianapolis, IN). After freezing at –80°C, cells were thawed and centrifuged at 20,000 g for 10 min to remove cellular debris. Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Nylon+ membrane (Novex, San Diego, CA) overnight at 100 mA. Membranes were probed in the primary antibody polyclonal antibody against the FLAG epitope (1:2000 dilution, Covance, CA) and the secondary antibody goat anti-rabbit IgG-HRP (1:5000 dilution, Santa Cruz, CA). PIERCE ECL Western Blotting Substrate (PIERCE, Rockford, IL) was used to visualize the secondary antibody.
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RESULTS |
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Zearalenone Activates PXR and ER
To investigate the regulation of nuclear receptors by zearalenone, we performed a series of reporter gene assays in CV-1 cells. As shown in Figure 1B, among all the nuclear receptors investigated, PXR and ER
are the most responsive. For the purpose of positive control, all nuclear receptors were treated with their cognate prototypical ligands, except for hPXR.2, of which no ligand has been identified. As expected, each receptor was activated by the cognate ligand (Fig. 1C).
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Zearalenone's activity against PXR exhibits species-selective properties, with hPXR being more responsive when compared with mouse PXR. The PXR.2 splice variant was not activated by zearalenone. Comparison to the classic species-selective PXR ligands revealed that zearalenone activated hPXR with about 70% efficacy of that of rifampicin, a prototypical hPXR ligand, while it only had about 40% efficacy on mouse PXR when compared to pregnenolene 16
-carbonitrile (PCN), a prototypical rodent PXR ligand (Fig. 1D). Reporter gene assays using ER demonstrated that zearalenone activated ER with an efficacy comparable to that of the full agonist 17ß-estradiol. Full concentration-response analysis on PXR revealed that zearalenone was more potent and efficacious at activating hPXR when compared with mouse PXR (Fig. 1E).
Zearalenone Induces Endogenous Cytochrome P450 3A in a hPXR-Dependent Manner
To determine the extent to which zearalenone induces the expression of endogenous PXR-target genes in liver cells, we treated adenovirus-transduced HepG2 cells and performed real-time quantitative PCR analysis for CYP3A4, the prototypical PXR-target gene in human liver. Western blot analysis confirmed that adenoviral transduction resulted in the expression of the FLAG-tagged hPXR protein (Fig. 2A). Notably, zearalenone treatment induced CYP3A4 gene expression in HepG2 cells that were transduced with hPXR adenovirus, while this effect was nearly absent in the HepG2 cells that were transduced with the GFP (blank) adenovirus (Fig. 2B). To confirm the results from HepG2 cells and to determine the biological activity of zearalenone in a normal hepatic cellular environment, we also performed similar experiments in primary cultures of mouse hepatocytes that were isolated from PXR-KO mice. Western blot analysis demonstrated the expression of the FLAG-tagged hPXR protein in primary mouse hepatocytes after adenoviral transduction (Fig. 3A). As shown in Figure 3B, in PXR-KO mouse hepatocytes that were transduced with the hPXR adenovirus, zearalenone induced the gene expression of Cyp3a11, the prototypical PXR-target gene in mouse liver. In primary cultures of PXR-KO mouse hepatocytes that were transduced with the blank virus, zearalenone had no effect on the Cyp3a11 gene expression. Taken together, these data strongly suggest that the induction of CYP3A by zearalenone is mediated by PXR.
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Zearalenone Modulates the Interaction between PXR and Cofactors
The interaction between nuclear receptor corepressor proteins and nuclear receptors represses nuclear receptor activity in cells. On the other hand, the interaction between SRC proteins and nuclear receptors enhances nuclear receptor activity in cells. To investigate the molecular mechanism whereby zearalenone activates hPXR, we sought to determine the extent to which it differentially modulates the PXR-cofactor protein-protein interaction using the mammalian two-hybrid system. Cultures of CV-1 cells were transfected with the expression vector encoding the receptor-interacting domain from cofactor proteins fused to GAL4 DNA-binding domain and the expression vector encoding VP16-tagged hPXR together with the GAL4-responsive luciferase reporter gene pFR-LUC (Fig. 4A). As shown in Figure 4B, zearalenone efficaciously displaced the corepressor N-CoR from PXR. Similarly, zearalenone efficaciously recruited the coactivators SRC-1, PBP, and GRIP1 to PXR (Fig. 4C–E). These data suggest that zearalenone likely binds PXR and serves as a direct ligand for this receptor.
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DISCUSSION
In this study, we have demonstrated that zearalenone activates the nuclear receptor PXR and induces the expression of the drug-metabolizing enzyme CYP3A4, a hepatic monooxygenase involved in the metabolism of about 60% of clinically used drugs (Guengerich, 1999
). PXR activation has been shown by multiple groups to represent the molecular basis for drug-drug interactions and herb-drug interactions. Full-concentration analysis revealed that zearalenone activated hPXR at low micromolar levels. Although currently it is not known how high zearalenone concentration can reach in humans exposed to zearalenone-contaminated food, values up to 140 mg/kg and more of zearalenone in agricultural products have been reported (Schoental, 1983). In addition, high oral bioavailability (28.1%) of zearalenone has been reported in rats (Mallis et al., 2003). Thus, it is likely that zearalenone activates PXR in humans under certain circumstances. Because PXR regulates many drug-metabolizing enzymes and drug transporters in human liver and intestine, activation of PXR by zearalenone likely promotes the metabolism and elimination of many other coadministered drugs either through metabolism- or transport-mediated mechanism or both. Therefore, our findings indicate that the consequences of PXR activation potentially extends from drug-drug and herb-drug interactions to food-drug interactions. Moreover, further work to investigate zearalenone-drug interactions in vivo is warranted.
In addition to drug-metabolizing enzymes, PXR also regulates the 25-hydroxyvitamin D3-24-hydroxylase (CYP24), a mitochondrial enzyme responsible for inactivating vitamin D metabolites (Pascussi et al., 2005). Therefore, drugs that activate PXR have the potential to cause vitamin D deficiency and eventually osteomalacia and osteoporosis. Our findings suggest that the potential of long-term exposure to zearalenone to cause osteomalacia and osteoporosis is worth further investigation.
At the molecular level, agonist ligands bind to nuclear receptors and cause conformational changes in the receptor that favor the release of corepressor proteins and recruitment of coactivator proteins. The corepressor proteins repress the activity of nuclear receptors by recruiting the histone deacetylase complex (Alland et al., 1997). On the other hand, the coactivators either contain endogenous histone acetyltransferase (HAT) activity or help recruit HAT-containing components to nuclear receptors (Puigserver et al., 1999; Spencer et al., 1997). Our mammalian two-hybrid analysis demonstrates that zearalenone activates PXR by displacing the corepressor protein N-CoR and recruiting the coactivators SRC-1, PBP, and GRIP1, suggesting that zearalenone most likely directly binds to PXR and acts as classical nuclear receptor ligand.
In vitro studies of hepatic gene expression are often performed in primary hepatocytes. However, human hepatocytes are not readily available. On the other hand, studies performed in rodent hepatocytes are hard to translate to humans due to the existence of species-specific differences. For example, rifampicin is a potent inducer of CYP3A gene expression in human hepatocytes, but it has little effect on CYP3A gene expression in mouse hepatocytes, while the synthetic steroid PCN strongly induces CYP3A gene expression in mouse hepatocytes but not in human hepatocytes. Recent studies have revealed that this striking species-specific difference between humans and mice is due to the evolutionary divergence of the PXR ligand-binding domain in these two species (Kliewer, 2003; Xie et al., 2000). The development of an adenoviral-mediated expression system to deliver functional FLAG-tagged hPXR protein to cultured cell models represents a potentially important tool. This novel tool can be used to quickly and reliably characterize the extent to which new drug candidate molecules activate this clinically important and biologically relevant signaling pathway. Our studies using this expression system to deliver hPXR to PXR-KO cultures of mouse hepatocytes demonstrate that this model responds to PXR activators in a way similar to human hepatocytes. Therefore, this novel cultured hepatocyte "humanized" mouse model will likely prove useful in the studies of the human xenobiotic response and may serve as a valuable predictor of potential drug interactions in hepatocytes.
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ACKNOWLEDGMENTS |
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We thank Dan Brobst for his technical assistance throughout this project. We thank Dr. Daniel Kelly for Ad-GFP construct. We thank Dr. Michael Stallcup for pGAD424-GRIP1 and Dr. Barry Forman for GAL4-N-CoR, GAL4-SRC-1, and GAL4-PBP. This work was supported by the National Institutes of Health grant 1R01DK068443-01A1.
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