Effects of Pharmaceuticals on the Expression of ... - ACS Publications

May 4, 2012 - Detoxification in a Carp Primary Hepatocyte Model. Jenna Corcoran, ... common to the genes involved in drug metabolism and transportatio...
0 downloads 0 Views 361KB Size
Download PDF
Article pubs.acs.org/est

Effects of Pharmaceuticals on the Expression of Genes Involved in Detoxification in a Carp Primary Hepatocyte Model Jenna Corcoran,†,* Anke Lange,† Matthew J. Winter,‡ and Charles R. Tyler†,* †

University of Exeter, Biosciences, College of Life & Environmental Sciences, Exeter, United Kingdom AstraZeneca Safety, Health and Environment, Brixham Environmental Laboratory, Freshwater Quarry, Brixham, United Kingdom

‡

S Supporting Information *

ABSTRACT: Fish in many surface freshwaters are exposed to a range of pharmaceuticals via wastewater treatment works effluent discharges. In mammals the pregnane X receptor (PXR) plays a key role in the regulation of a suite of genes involved in drug biotransformation, but information on the role of this response pathway in fish is limited. Here we investigated the effects of exposure of carp (Cyprinus carpio) primary hepatocytes to the human PXR agonist rifampicin (RIF) on expression of target genes involved in phase I (cyp2k, cyp3a) and phase II (gstα, gstπ) drug metabolism and drug transporters mdr1 and mrp2. RIF induced expression of all target genes measured and the PXR antagonist ketoconazole (KET) inhibited responses of cyp2k and cyp3a. Exposure of the primary carp hepatocytes to the pharmaceuticals ibuprofen (IBU), clotrimazole (CTZ), clofibric acid (CFA) and propranolol (PRP), found responses to IBU and CFA, but not CTZ or PRP. This is in contrast with mammals, where CTZ is a potent PXR-agonist. Collectively our data indicate potential PXR involvement in regulating selected genes involved in drug metabolism in fish, but suggest some divergence in the regulation pathways with those in mammals. The carp primary hepatocyte model serves as a useful system for screening for responses in these target genes involved in drug metabolism.

■

INTRODUCTION Fish can be exposed to a wide range of pollutants, including pharmaceutical drugs, many of which are commonly detected in surface waters and wastewater treatment work effluents.1 There has been significant research on the effects of selected pharmaceuticals in fish in recent years (reviewed in ref 1), but studies on chronic exposure effects to environmentally relevant concentrations, mixture effects, and mechanism of action in fish are lacking. Understanding pathways of action of pharmaceuticals will help identify biomarkers of exposure and effect for both in situ laboratory studies and studies on wild populations. Fish combat exposure to pollutants through a complex chemical defense system of enzymes and transport proteins capable of biotransformation and subsequent elimination of many such compounds from the body. Central to this system © 2012 American Chemical Society

are a number of nuclear receptors, which are ligand-activated transcription factors with important roles in regulating the metabolism and disposition of both endogenous and xenobiotic compounds.2 In humans, the pregnane X receptor (PXR; nuclear receptor subfamily 1, group I, member 2; NR1I2) is fundamental to drug metabolism, and a growing list of ligands has been identified in mammals.3 The PXR in mammals is most notably associated with regulation of phase I metabolism, controlling the phase I enzyme cytochrome P4503a4 (CYP3A4), implicated in the metabolism of over 60% clinically used human drugs4 and the biotransformation of a wide range of structurally Received: Revised: Accepted: Published: 6306

February 8, 2012 April 30, 2012 May 4, 2012 May 4, 2012 dx.doi.org/10.1021/es3005305 | Environ. Sci. Technol. 2012, 46, 6306−6314

Environmental Science & Technology

Article

in benzocaine (ethyl 4 amino-benzoate; 100 g L−1 ethanol, dissolved to ∼500 mg L−1 tank water) and injected with heparin (1000 units) before commencing the cell isolation procedure. The isolation of hepatocytes was performed using a two-step collagenase perfusion technique, carried out at room temperature in situ, using a protocol described previously18 and in accordance with the UK Animals (Scientific Procedures) Act, 1986. A detailed description of the method is included in the Supporting Information (SI). Briefly, the liver was perfused with a series of HEPES-buffered Hank’s solutions and digested by addition of collagenase D (Roche Diagnostics, Switzerland); the liver was then dissected from the body cavity, hepatocytes isolated by low speed centrifugation and the resultant pellet suspended in M199 culture medium. Primary Hepatocyte Culture. Hepatocytes were seeded at a density of 1.5 × 106 cells mL−1 in M199 culture medium (supplemented with HEPES, NaHCO3, CaCl2, fetal bovine serum, L-glutamine, penicillin, and streptomycin) and cultured in a humidified atmosphere at 20 °C. Following 24 h culture without exposure to chemicals, hepatocytes were then exposed to the appropriate test compound for up to 72 h. At 24 h intervals 50% of the culture medium with appropriate test compound was replaced, with a maximum solvent concentration of 0.02%. Hepatocyte cultures were prepared from 13 fish with each treatment replicated for at least 4 separate cultures. A detailed protocol is included in the SI. Cell Viability Assessment. Cell viability was assessed throughout the culture period by measuring leakage of lactate dehydrogenase (LDH) from hepatocytes into the culture medium using the CytoTox 96 nonradioactive cytotoxicity assay (Promega, Southampton, UK). Maximal LDH from completely lysed cells was determined and cell viability in the cultures was expressed as a percentage of complete viability (i.e., no LDH leakage). RNA Extraction, Reverse Transcription, and Primer Design. Following the appropriate exposure period, the culture medium was removed and the hepatocytes lysed by addition of Tri-Reagent,22 then snap frozen in liquid nitrogen and stored at −80 °C, for subsequent analysis of target mRNA expression. Total RNA was isolated using Tri-Reagent22 following the manufacturer’s instructions, and the amount quantified using a NanoDrop spectrophotometer. RNA quality was determined both by electrophoresis on an ethidium bromide-stained 1.5% agarose gel and through the measurement of A260/A280 ratio. One μg RQ1 DNase treated (Promega, Southampton, UK) total RNA was subsequently reverse transcribed to cDNA using random hexamers (Eurofins MWG Operon, Ebersburg, Germany) and MMLV reverse transcriptase (Promega), according to the protocol described previously. 23 Gene sequences were not available for common carp cyp2k and cyp3a, and so partial sequences were first established for these genes. Details of primers used for obtaining cyp2k and cyp3a sequences and for real time quantitative polymerase chain reaction (RT-qPCR) are shown in SI Table S1. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) for Target Gene mRNA Analysis. RT-qPCR was carried out for each of the target genes with the appropriate samples, in triplicate, as described previously.23 Briefly, primer pairs were optimized for annealing temperature (Ta), specificity confirmed by melt curve analysis, and the detection range, linearity and amplification efficiency (E) established using serial dilutions of carp liver cDNA. RT-qPCR was carried out using Absolute QPCR SYBR Green Fluorescein mix (ABgene,

unrelated substrates. The mammalian PXR also regulates a series of other genes involved in phase I metabolism, including members of cyp1a, cyp2b, and cyp2c families,5 and enzymes in phase II metabolism, including glutathione-S-transferases (GSTs), UDP-glucuronosyltransferases (UGTs) and sulfotransferases (STs),6 which catalyze the conjugation, and therefore further detoxification, of phase I metabolites. In mammals PXR plays a further role in the regulation of drug transporters such as P-glycoprotein (P-gp, Mdr1, or abcb1), several multidrug resistance related proteins (mrps) and organic anion transporter protein 2 (oatp2). In fish, in contrast, there is little information on the PXR, the downstream genes and their roles in the metabolism and transport of drugs. An important characteristic common to the genes involved in drug metabolism and transportation in mammals is that they can be induced following exposure to their substrates, as well as other structurally unrelated compounds.7 The modulation of the PXR and downstream genes in this way may therefore potentially affect the pharmacokinetics, clearance, and homeostatic balance of a wide range of both endogenous and xenobiotic compounds in the fish.8,9 Such responses, in turn, could impact on the ability of the fish to cope with those exposures; indeed in humans, modulation of cyp3a4 expression is responsible for a range of potentially harmful drug−drug interactions.10 Certain isoforms of the cytochrome P450 (CYP) enzymes have been shown to be responsive to several pharmaceuticals in fish11 and as such, induction of these and other PXR-associated genes may be a potentially useful tool for biomonitoring of this type of environmental contamination.12 Consistent with its role in regulating xenobiotic-metabolizing enzymes, the PXR is primarily expressed in the liver.13 Here we employed common carp (Cyprinus carpio) primary hepatocytes to investigate the expression of a suite of target genes known to be regulated via the PXR in mammals, specifically, cyp3a, cyp2k, gstα, gstπ, mdr1, and mrp2. In humans, primary hepatocytes are taken as the standard model for in vitro testing of drugs, and are indeed the only in vitro model that can produce a metabolic profile for a drug similar to that found in vivo.14 Similarly, primary hepatocytes have previously proven to be a sensitive and reproducible model with which to study biotransformation in fish in vitro.15−20 In the present study carp hepatocytes were exposed to rifampicin (RIF), a potent PXR agonist in mammals, as a positive control and to a further four test pharmaceuticals from different therapeutic groups; ibuprofen (IBU, nonsteroidal anti-inflammatory drug, NSAID), clotrimazole (CTZ, azole antifungal), propranolol (PRP, β-blocker), and clofibric acid (CFA, fibrate), all of which are found to be relatively prevalent in the aquatic environment. We further investigated inhibition of the apparent PXR-mediated induction of cyp2k and cyp3a by coexposure of cells to RIF and ketoconazole (KET), a PXR antagonist in mammals.21

■

MATERIALS AND METHODS Animals. Adult, mixed-sex common carp (Cyprinus carpio) weighing approximately 200 g, obtained from Priory Fisheries (Devon, UK) were held in aerated tanks in a flow-through water system, at 12 ± 1 °C, maintained under a 12 h daylight cycle and fed daily ad libitum with commercial pellets (Nishikoi, UK). Carp were acclimated for 6 weeks prior to cell isolation and feeding was withheld 1 day before cell isolation. Hepatocyte Isolation. All chemicals and reagents were obtained from Sigma-Aldrich (Poole, UK) unless stated otherwise. Fish were anaesthetized terminally by immersion 6307

dx.doi.org/10.1021/es3005305 | Environ. Sci. Technol. 2012, 46, 6306−6314

Environmental Science & Technology

Article

Figure 1. Expression of (a) cyp2k and (b) cyp3a in primary hepatocytes exposed to RIF [10 μM] (solid line) and control groups (dashed line) over the culture duration. Expression is shown as fold difference relative to mean control. N = 4 cultures from separate fish, with four replicates per culture for each treatment at each time point. Error bars represent standard errors. The asterisks indicate the first time point of significant difference between control and RIF [10 μM] treatment groups and thereafter differences continue to be significant (p < 0.05).

Figure 2. Expression of (a) cyp2k and (b) cyp3a on coexposure to RIF and KET for 72 h. Expression is shown as fold difference relative to mean control. N = 4 cultures from separate fish with four replicates from each culture for each treatment; error bars represent standard error. Statistically significant differences in fold-changes in relative gene expression levels between treatment groups are denoted by different letters.

■

Epsom, UK,), with an initial activation step of 95 °C for 15 min followed by 40−50 cycles of denaturation (95 °C, 10 s) and annealing (appropriate Ta, 45 s) and final melt curve analysis. Ribosomal protein 8 (rpl8) was used as a “housekeeping” gene, to normalize the target gene expression, using a development of efficiency correlated relative quantification as described previously, 23 as it was found not to alter following exposure to any of the test chemicals (p > 0.05 in all cases; see SI Figure S1). Data Analysis. Throughout this paper data are presented as mean ± standard error of the mean. All statistical analyses were carried out using SigmaPlot software (Systat Software, Inc., Chicago, IL). Data were tested for normality/equal variance and log transformed if necessary. Effects of test compounds on levels of gene expression and cell viability were determined using one-way ANOVA followed by Fisher LSD multi comparison procedure, where appropriate. Factorial ANOVA was used to test for significant interaction in the coexposure treatments. Where data did not meet assumptions of normality and/or homogeneity of variance, data were analyzed using Kruskal−Wallis one-way ANOVA on ranks followed by Dunn’s post hoc analysis. In all cases, p < 0.05 was considered statistically significant.

RESULTS Cell Viability. Mean viability of hepatocytes in controls was greater than 90%, with no change in viability over the culture period (up to 96 h), based on seven separate cultures (SI Figure S2A). There was no significant effect of the any of the test chemicals, at the concentrations tested on cell viability, with the exception for CTZ, where at the highest test concentration (100 μM) cell viability was 71% after 24 h, 60% at 72 h, and significantly lower than in controls across all time points (SI Figure S2B). No molecular analyses were conducted on the hepatocytes derived from this chemical dosing for CTZ. Sequence Identification of cyp2k and cyp3a. We identified the partial sequences of cyp2k (629 bp) and cyp3a (654 bp) isoforms in common carp (GenBank accession numbers: GU19996 and GU19997, respectively) which demonstrated a high degree of identity with these gene sequences in other teleosts (in particular cyprinids); for cyp2k up to 79% and for cyp3a up to 88% sequence similarity. Cyp Expression over Culture Duration. Expression of cyp2k and cyp3a were first measured in hepatocytes exposed to 10 μM RIF and in controls over a 72 h time-course (Figure 1). Cyp2k expression in cells exposed to RIF increased over the 6308

dx.doi.org/10.1021/es3005305 | Environ. Sci. Technol. 2012, 46, 6306−6314

Environmental Science & Technology

Article

Figure 3. Target gene expression relative to mean controls in hepatocytes exposed to pharmaceuticals (RIF, IBU, CFA, CTZ, and PRP) at three different concentrations [0.01 (white bars), 1 (gray bars), 100 μM (black bars)] and controls (striped bar) for 72 h. Target genes are (a) cyp2k, (b) cyp3a, (c) gstα, (d) gstπ, (e) mdr1, and (f) mrp2. N = 4 or 5 cultures from separate fish, with at least two replicates per fish for each treatment. Error bars represent standard error. Note values are expressed on a logarithmic scale. Asterisk above bar indicates significant difference to the control group (p < 0.05).

was dependent on the level of KET), whereas cyp3a expression was significantly reduced only at concentrations of 1 and 10 μM KET and there was no statistically significant interaction. Exposure of hepatocytes to 10 μM KET alone, significantly induced expression of cyp2k, but not cyp3a, relative to the control. Expression of Target Genes on Exposure to Test Pharmaceuticals. Expression of cyp2k was significantly induced in hepatocytes exposed to all concentrations of the positive control RIF, with up to 20-fold induction at 100 μM (Figure 3). Cyp2k expression was also significantly induced in hepatocytes exposed to IBU at all test concentrations and by up to 14-fold at 100 μM, and CFA at the higher two concentrations (up to14-fold induction at 100 μM). CTZ significantly down-regulated cyp2k expression by 3-fold at 1 μM but had no effect at 0.01 μM. PRP had no significant effect on cyp2k expression at any test concentration. Cyp3a expression was significantly induced on exposure to RIF at all test concentrations, up to 8-fold at 100 μM, by all concentrations of IBU, up to 3-fold at 100 μM, and by CFA at

duration of the culture, and was significantly greater compared with controls at 12 h exposure and thereafter, with a 21-fold induction at 72 h (Figure 1A). Similarly, cyp3a expression in cells exposed to RIF increased over the culture duration and was significantly elevated compared with controls at 24 h, and time points thereafter, with an 8-fold induction at 72 h (Figure 1B). Expression of each target gene did not change over the entire culture period in control hepatocytes (p > 0.05), indicating stable basal expression of both cyp isoforms. Inhibition of the cyp2k- and cyp3a-Induction Responses. Hepatocytes were coexposed to 10 μM RIF plus KET at four concentrations between 0.01 μM and 10 μM for 72 h, with hepatocytes exposed to 10 μM RIF without KET taken as a positive control (Figure 2). Coexposure of hepatocytes to KET and RIF significantly reduced the expression of both cyp2k and cyp3a relative to the positive control. The induced expression of cyp2k in response to RIF was significantly reduced by the addition of even 0.01 μM KET and there was a significant interaction between the two compounds (i.e., the expression of cyp2k in cells exposed to RIF 6309

dx.doi.org/10.1021/es3005305 | Environ. Sci. Technol. 2012, 46, 6306−6314

Environmental Science & Technology

Article

RIF failed to significantly induce PXR activation in a rainbow trout reporter assay or corresponding cyp3a mRNA in vitro in rainbow trout cell line RTH-149.20 Similarly, RIF was not found to induce CYP3A protein levels in PLHC-1 cells,33 and collectively these findings suggest possible species differences in response pathways in fish. Cyp2k expression was highly responsive to RIF (up to a 22fold induction) suggesting regulation via the PXR. Cyp2k, similar to cyp3a, is a major constitutively expressed cyp gene in fish liver9 but there has been limited characterization of this isoform, and no previous report of induction by RIF. Cyp2k is not present in mammals, but it is thought to be analogous to the mammalian cyp2b subfamily34 which is inducible by RIF, but via activation of the constitutive androstane receptor (CAR),32 a nuclear receptor which is lacking in fish; it is thought that CAR appeared after the divergence of fish in evolution.35,36 In mammals, RIF is also known to induce the cyp2c isoforms, probably via the PXR.37 KET, an azole antifungal agent, is a well-known inhibitor of CYP3A enzyme activity.38 In mammals, KET also inhibits cyp3a at the transcriptional level, as it is an antagonist of the PXR.21 In agreement with this, the inductive responses of both cyp3a and cyp2k to RIF exposure were inhibited here in carp hepatocytes in a concentration-dependent manner supporting the theory of the PXR playing a role in both cyp3a and cyp2k modulation in fish. Furthermore, the data for the KET inhibition of induced cyp3a mRNA expression are comparable to results obtained in a human hepatocyte cell line (Fa2N-4), where KET inhibited RIF-induced cyp3a mRNA expression at concentrations between 2.5 and 40 μM.39 Conversely there are reports of KET inducing cyp3a expression in fish in vivo when administered alone40,41 (although CYP3A enzyme activity was inhibited) and in Fa2N-4 cells, KET was shown to activate the basal level transcriptional activity of PXR in the absence of a ligand (but drastically inhibited PXR activation in the presence of RIF).39 This would suggest that alone KET induces cyp3a expression, possibly via PXR activation, but acts as a competitive inhibitor in the presence of an activator such as RIF. Further studies are needed, however, to confirm this. Interestingly in our study, although cyp2k expression was induced marginally by exposure to 10 μM KET there was no significant increase in cyp3a expression above control levels in carp hepatocytes. Induction of Glutathione-S-Transferase by Rifampicin. GSTs are a family of detoxifying phase II enzymes that catalyze the conjugation of reduced glutathione to electrophilic centers on a wide range of substrates. This modifies endogenous compounds such as lipid peroxidases, as well as various xenobiotics, facilitating their dissolution into the aqueous cellular/extracellular matrix and ultimately their excretion from the body. As such, it makes sense that a xenobiotic compound induces the expression of these enzymes responsible for eliminating the reactive intermediates generated by phase I reactions, for example, those catalyzed by CYPs. We demonstrate here that gstα and gstπ isoforms are induced by 100 μM RIF, up to 5- and 3-fold compared with hepatocytes without treatment, respectively. This is comparable to a study on human primary hepatocytes where gstα and gstπ isoforms were induced 3- and 4-fold, respectively, on exposure to 33 μM RIF.42 The responses here of gstα and gstπ isoforms to RIF implicate possible involvement of the PXR, however GST regulation is complex.9 In mammals, although other regulatory pathways predominate in gst modulation, for example via the

the two highest exposure concentrations (up to 1.5-fold induction at 100 μM). CTZ down-regulated cyp3a expression by 2-fold at 1 μM, but with no significant effect at 0.01 μM. PRP had no effect on cyp3a expression at any of the test concentrations. Gstα expression was significantly induced in hepatocytes exposed to both 1 and 100 μM RIF and IBU up to 5- and 1.3fold inductions at 100 μM, respectively. CTZ significantly down-regulated expression of gstα by 1.4-fold at 1 μM, but had no significant effect at 0.01 μM. PRP or CFA had no significant effect on gstα expression at any concentration tested. Expression of gstπ was significantly induced on exposure to RIF and IBU at all test concentrations, up to levels of 3- , 1.5fold, respectively, at 100 μM; also on exposure to CFA at the higher two concentrations, up to 1.3-fold at 100 μM. CTZ and PRP had no significant effect on gstπ expression at any test concentration. Mdr1 expression was significantly induced in hepatocytes exposed to RIF at all concentrations, up to 6-fold at 100 μM. Mdr1 expression was also induced on exposure to 1 and 100 μM IBU, CFA, and PRP with 3-, 2-, and 1.5-fold inductions, respectively, at 100 μM. Mdr1 expression was down regulated on exposure to CTZ at both concentrations (1.5-fold decrease at 1 μM). Mrp2 expression was significantly induced in hepatocytes exposed to RIF at all concentrations, up to 2-fold at 100 μM. Expression of mrp2 was also induced on exposure to 1 and 100 μM IBU and CFA, and to 100 μM PRP, with up to 2-, 1.5-, and 1.5-fold inductions, respectively, at 100 μM. CTZ had no significant effect at either test concentration.

■

DISCUSSION The major objectives of this study were to investigate the expression of selected PXR-target genes involved in drug metabolism in common carp compared with that established for mammals and to assess the applicability of carp primary hepatocytes as a model for studying these end points. Primary cultures of mammalian hepatocytes have been shown to serve as a sensitive model for analyzing the regulation of CYP modulation by drugs and other chemicals, maintaining constitutive CYP activity at levels close to those in freshly isolated hepatocytes.14,24,25 Here for carp hepatocytes, we demonstrate good cell viability and moreover, consistent constitutive expression of the two target cyp genes (cyp2k, cyp3a) examined for the duration of the culture period, indicating the suitability of this model for studies on expression of the selected genes. Cyp Induction by Rifampicin. RIF is a well characterized human PXR agonist commonly used as a positive control for PXR activation and cyp3a induction.26,27 That said, there are conflicting reports on PXR activation by RIF in other mammalian species (reviewed in ref 28). Cyp3a induction in fish is thought to be predominantly regulated by transcriptional activation via PXR, with full PXR (NR1I2) sequences identified in zebrafish (Danio rerio)30,31 and rainbow trout (Oncorhynchus mykiss)20 and partial sequence in fathead minnow (Pimephales promelas).29 Here, exposure to RIF induced cyp3a in a concentration-dependent manner with a maximal induction of 8-fold compared with controls at 100 μM. The RIF-induction of cyp3a mRNA expression in carp hepatocytes supports other studies in cyprinid fish, both in vivo, and in vitro using fish cell lines, where a dose-dependent increase in CYP3A-related enzyme activity has been reported.11,32 In contrast with this, 6310

dx.doi.org/10.1021/es3005305 | Environ. Sci. Technol. 2012, 46, 6306−6314

Environmental Science & Technology

Article

mechanism underlying this response is not clear. There are no reports in mammals or in fish of CFA being a PXR ligand. Instead CFA is a well-known ligand of a different nuclear receptor, the peroxisome proliferator-activated receptors (PPARα and PPARγ) which most notably mediate cyp4. Neither PPAR isoform is directly associated with the regulation of the genes measured in the present study, and so the observed results are difficult to explain. In silico analysis has suggested that human PXR could be transcriptionally activated by PPARα binding a PPAR-response element (PPRE) in the promoter region of the pxr gene.66 Thus apparent inducers of PXR (e.g., clofibrate) which are not direct ligands, may activate PXR indirectly via this mechanism. Indeed, in rats clofibrate has been shown to induce pxr67 and furthermore to induce cyp2b and cyp3a mRNAs, proteins and enzyme activities.68 Target Gene Analysis on Exposure to Ibuprofen. All genes analyzed were significantly induced on exposure to 1 and 100 μM IBU and some to a relatively high level (e.g., cyp2k was induced 14-fold at the highest test concentration, relative to unstimulated hepatocytes). This is surprising as IBU has shown no activation of the human PXR in reporter gene assays69 and moreover, in humans IBU is approved as having no known cyp3a4 induction liability with regard to drug−drug interactions. There are no previous reports of PXR activation in response to IBU in fish, however complementary with our results, IBU showed a slight inductive effect on mdr1 expression in PLHC-1 cells, (although an inhibitory effect was seen on Pgp transport function).60 IBU has also previously been shown to inhibit CYP2K-like and CYP2M-like enzyme activity in carp in vitro.70 IBU induces gstα/π in rats71 although the mechanism is unclear, and has also been shown to be a PPARα/γ ligand in murine cell line C3H10T1/2.72 so it is interesting to speculate whether these genes may be induced via the same mechanism described above for CFA. Target Gene Analysis on Exposure to Propranolol. We found PRP had no significant effect on the genes analyzed involved in phase I or II drug metabolism at any concentration tested, however expression of the drug transporter genes mdr and mrp2 were significantly induced (1.5-fold in both cases) at 100 μM in the carp hepatocytes. This corresponds with a previous study in PLHC-1 cells where exposure to PRP induced mdr1 expression (by approximately 2-fold) although was not related to P-gp activity.60 There are, to our knowledge, no data linking PRP with the PXR or downstream target genes in mammals or in fish. Accordingly, one study which screened 170 pharmaceuticals for hPXR activation found that PRP failed to activate this receptor.73 Taking this into account, with our own results, it would seem reasonable to suggest that the observed induction of the mdr1 is not via the PXR. In summary, the induction of several important genes involved in xenobiotic metabolism and elimination by RIF suggests that the PXR is potentially a key player in regulating the expression of these genes in fish, as has been established for mammals. In carp primary hepatocytes responses of metabolic enzyme systems and drug transporters (albeit measured through gene expression alone) to drug exposures were found to be equally sensitive as that reported in mammalian systems, highlighting their potential vulnerability to pharmaceuticals discharged into aquatic environments. Screening of pharmaceuticals from different therapeutic classes showed some commonalities, but also differences in the patterns of expression of the target genes suggesting some evolutionary divergence in the regulatory systems. This emphasizes that responses in

Nrf-2/antioxidant response element (ARE), the glucocorticoid receptor (GR) or the aryl hydrocarbon receptor (AhR) pathways43 several gst isozymes have been identified as putative PXR target genes from DNA microarray analysis44 and as noted above, there are reports of gsts being induced by RIF.42 Studies of the molecular mechanism of gst induction in fish are lacking, but it has been suggested previously that gst gene expression and enzyme activity also respond to ligands of both the AhR45,46 and the ARE47,48 as in mammals. Induction of Drug Transporters by Rifampicin. Pglycoprotein (P-gp) encoded by the mdr1 or abcb1 gene, and multidrug resistance-associated protein 2 (encoded by mrp2 or abcc2) are well-characterized members of the superfamily of ATP-binding cassettes (ABC) transporters, which function as pumps mediating the efflux of various molecules across cellular membranes. These ABC transporters have an essential role in eliminating toxic compounds from the body, and likely evolved as a defense mechanism against harmful substances.49 This efflux is also the principal mechanism by which many cancers develop resistance to chemotherapy drugs.50 P-gp and MRP2 transport a broad range of compounds, including drugs and conjugated drug metabolites and have been shown to be induced to higher levels of expression by a number of compounds. In mammals, it is now known that this regulation is predominantly via the PXR, and also the CAR. 49 Furthermore, mdr1 is thought to be functionally linked with cyp3a; showing broad overlaps in substrate specificity and are often induced together.51 Accordingly, there are numerous studies in mammals demonstrating RIF as an inducer of both mdr1 and mrp2.51−57 In fish, mdr1 and mrp2 genes have been identified in several species, including common carp and zebrafish, as well as winter flounder (Pseudopleuronectes americanus), killifish (Fundulus heteroclitus), sheepshead minnow (Cyprinodon variegatus) and channel catfish (Ictalurus punctatus)58,59 and in cells of the teleost hepatocellular carcinoma cell line PLHC-1.60 Analysis of the zebrafish genome has revealed 77% of human ABC transporters have an ortholog in zebrafish.61 However, there is little data on the regulation of these genes in fish, and to our knowledge this is the first study to show induction of these genes by RIF, demonstrating a possible role for the PXR in the regulation of these genes in carp. Previously, the PXR agonist pregnenolone 16α-carboninitrile has been shown to induce expression of mdr1, alongside cyp3a and pxr in zebrafish, whereas CTZ or nifedipine, were both shown to be PXR agonists in mammals, had no significant effect on expression of these genes.62 Target Gene Analysis on Exposure to Clotrimazole. Clotrimazole (CTZ), an azole antifungal, is a highly potent ligand of the human PXR, and accordingly has been demonstrated to induce expression of several PXR associated target genes including cyp3a4,63 mdr151 and mrp2.64 Although CTZ has also been suggested to be a ligand of the PXR in fish based on an in vitro study in zebrafish,30 here we found no induction of any of the analyzed genes by CTZ, which is in agreement with two other studies in fish, one on cyp3a and mdr1 in zebrafish62 and the other on a cyp3a isoform in sea bass (Dicentrachus labrax).65 Interestingly, in our study we show a slight, but significant down-regulation of cyp2k, cyp3a, gstα, and mdr1 expression relative to the control group, on exposure to 1 μM CTZ. Target Gene Analysis on Exposure to Clofibric acid. All target genes, with the exception of gstα, were significantly induced on exposure to 1 and 100 μM CFA although the 6311

dx.doi.org/10.1021/es3005305 | Environ. Sci. Technol. 2012, 46, 6306−6314

Environmental Science & Technology

Article

fishes. In The Toxicology of Fishes; Di Giulio, R. T., Hinton, D. E., Eds.; CRC Press: Boca Raton, FL, 2008; pp 153−234. (10) Thummel, K. E.; Wilkinson, G. R. In vitro and in vivo drug interactions involving human CYP3A. Annu. Rev. Pharmacol. Toxicol. 1998, 38, 389−430. (11) Li, D.; Yang, X.-L.; Zhang, S.-J.; Lin, M.; Yu, W.-J.; Hu, K. Effects of mammalian CYP3A inducers on CYP3A-related enzyme activities in grass carp (Ctenopharyngodon idellus): Possible implications for the establishment of a fish CYP3A induction model. Comp. Biochem. Physiol. C 2008, 147, 17−29. (12) Christen, V.; Caminada, D.; Arand, M.; Fent, K. Identification of a CYP3A form (CYP3A126) in fathead minnow (Pimephales promelas) and characterisation of putative CYP3A enzyme activity. Anal. Bioanal. Chem. 2010, 396, 585−595. (13) Williams, S. N.; Dunham, E.; Bradfield, C. A. Induction of cytochrome P450 enzymes. In Cytochrome P450: Structure, metabolism and biochemistry, 3E; Oritz de Montellano, P. R., Ed.; Springer: New York, 2005; pp 323−346. (14) Gomez-Lechon, M. J.; Donato, M. T.; Castell, J. V.; Jover, R. Human hepatocytes as a tool for studying toxicity and drug metabolism. Curr. Drug Metab. 2003, 4, 292−312. (15) Laville, N.; Ait-Aissa, S.; Gomez, E.; Casellas, C.; Porcher, J. M. Effects of human pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes. Toxicology 2004, 196, 41−55. (16) Segner, H.; Cravedi, J. P. Metabolic activity in primary cultures of fish hepatocytes. ATLA, Altern. Lab. Anim. 2001, 29, 251−257. (17) Pesonen, M.; Andersson, T. B. Fish primary hepatocyte culture; An important model for xenobiotic metabolism and toxicity studies. Aquat. Toxicol. 1997, 37, 253−267. (18) Bickley, L. K.; Lange, A.; Winter, M. J.; Tyler, C. R. Evaluation of a carp primary hepatocyte culture system for screening chemicals for oestrogenic activity. Aquat. Toxicol. 2009, 94, 195−203. (19) Sadar, M. D.; Andersson, T. B. Regulation of cytochrome P450 in a primary culture of rainbow trout hepatocytes. In Vitro Cell. Dev. Biol. Anim. 2001, 37 (3), 180−184. (20) Wassmur, B.; Grans, J.; Kling, P.; Celander, M. C. Interactions of pharmaceuticals and other xenobiotics on hepatic pregnane X receptor and cytochrome P450 3A signaling pathway in rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 2010, 100, 91−100. (21) Huang, H.; Wang, H.; Sinz, M.; Zoeckler, M.; Staudinger, J.; Redinbo, M. R.; Teotico, D. G.; Locker, J.; Kalpana, G. V.; Mani, S. Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole. Oncogene 2007, 26, 258−268. (22) Chomczynski, P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 1993, 15, 532−537. (23) Filby, A. L.; Tyler, C. R. Molecular characterization of estrogen receptors 1, 2a, and 2b and their tissue and ontogenic expression profiles in fathead minnow (Pimephales promelas). Biol. Reprod. 2005, 73, 648−662. (24) LeCluyse, E.; Madan, A.; Hamilton, G.; Carroll, K.; DeHaan, R.; Parkinson, A. Expression and regulation of cytochrome P450 enzymes in primary cultures of human hepatocytes. J. Biochem. Mol. Toxicol. 2000, 14, 177−188. (25) Brown, L. A.; Arterburn, L. M.; Miller, A. P.; Cowger, N. L.; Hartley, S. M.; Andrews, A.; Silber, P. M.; Li, A. P. Maintenance of liver functions in rat hepatocytes cultured as spheroids in a rotating wall vessel. In Vitro Cell. Dev.-An. 2003, 39, 13−20. (26) Kliewer, S. A.; Goodwin, B.; Sillson, T. M. The nuclear pregnane X receptor: A key regulator of xenobiotic metabolism. Endocrinol. Rev. 2002, 23, 687−702. (27) Bertilsson, G.; Heidrich, J.; Svensson, K.; Asman, M.; Jendeberg, L.; Sydow-Backman, M.; Ohlsson, R.; Postlind, H. Blomquist, p.; Berkenstam, A. Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12208−12213. (28) LeCluyse, E. L. Pregnane X receptor: Molecular basis for species differences in CYP3A induction by xenobiotics. Chem.-Biol. Interact. 2001, 134 (3), 283−289.

mammals to pharmaceutical compounds may not necessarily be predictive of effects to fish and other species, reinforcing the need for testing of pharmaceutical effects across a range of wildlife species for effective ecological risk assessment. Fish (here carp) primary hepatocytes combined with gene expression studies provide a useful model for screening of pharmaceuticals for their effect pathways in fish.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional details and description of some of the methods used as well as supporting figures and tables referred to in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone +44 (0)1392 264450; fax 44 (0)1392 263700; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Jan Shears and Mike Wetherell (University of Exeter) for their technical support and Don Mitchell (University of Exeter) for discussions on statistical analysis. J. Corcoran was funded by a Biotechnology and Biological Sciences Research Council Case studentship supported by AstraZeneca UK Ltd. (grant reference BB/G529332). A. Lange was supported by grants from the Natural Environmental Research Council (NE/ D002818/1 and NE/E016634/1) and DEFRA awarded to CRT. AstraZeneca Ltd. develops, produces, and markets a wide range of pharmaceutical agents.

■

REFERENCES

(1) Corcoran, J.; Winter, M. J.; Tyler, C. R. Pharmaceuticals in the aquatic environment: A critical review of the evidence for health effects in fish. Crit. Rev. Toxicol. 2010, 40, 287−304. (2) Janosek, J.; Hilscherová, K.; Bláha, L.; Holoubek, I. Environmental xenobiotics and nuclear receptorsInteractions, effects and in vitro assessment. Toxicol. In Vitro 2006, 20 (1), 18−37. (3) Lyer, M.; Reschly, E. J.; Krasowski, M. D. Functional evolution of the pregnane X receptor. Expert Opin. Drug Metab. Toxicol. 2006, 2, 381−397. (4) Maurel, P., The CYP3A family. In Cytochromes P450; Ionnides, C., Ed.; CRC Press: Boca Raton, FL, 1996; pp 241−270. (5) Shukla, S. J.; Nguyen, D.-T.; MacArthur, R.; Simeonov, A.; Frazee, W. J.; Hallis, T. M.; Marks, B. D.; Singh, U.; Eliason, H. C.; Printen, J.; Austin, C. P.; Inglese, J.; Auld, D. S. Identification of Pregnane X Receptor ligands using time-resolved fluorescence energy transfer and quantitative high-throughput screening. Assay Drug Dev. Technol. 2009, 7 (21), 143−169. (6) Tien, E. S.; Negishi, M. Nuclear receptors CAR and PXR in the regulation of hepatic metabolism. Xenobiotica 2006, 36 (10−11), 1152−1163. (7) Lehmann, J. M.; McKee, D. D.; Watson, M. A.; Willson, T. M.; Moore, J. T.; Kliewer, S. A. The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J. Clin. Invest. 1998, 102, 1016−1023. (8) Zhang, B.; Xie, W.; Krasowski, M. D. PXR: A xenobiotic receptor of diverse function implicated in pharmacogenetics. Pharmacogenomics 2008, 9 (11), 1695−1709. (9) Schlenk, D.; Celander, M.; Gallagher, E. P.; George, S.; James, M.; Kullman, S. W.; van den Hurk, P.; Willett, K., Biotransformation in 6312

dx.doi.org/10.1021/es3005305 | Environ. Sci. Technol. 2012, 46, 6306−6314

Environmental Science & Technology

Article

(29) Milnes, M. R.; Garcia, A.; Grossman, E.; Grun, F.; Shiotsugu, J.; Tabb, M. M.; Kawashima, Y.; Katsu, Y.; Watanabe, H.; Iguchi, T.; Blumberg, B. Activation of steroid and xenobiotic receptor (SXR, NR1I2) and its orthologs in laboratory, toxicologic, and genome model species. Environ. Health. Perspect. 2008, 116 (7), 880−885. (30) Moore, L. B.; Maglich, J. M.; McKee, D. D.; Wisely, B.; Willson, T. M.; Kliewer, S. A.; Lambert, M. H.; Moore, J. T. Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors. Mol. Endocrinol. 2002, 16, 977−986. (31) Bainy, A. C. D.; Stegeman, J. J. Cloning and identification of a full length pregnane X receptor and expression in vivo in zebrafish (Danio rerio). Mar. Environ. Res. 2004, 58, 133−134. (32) Christen, V.; Oggier, D. M.; Fent, K. A microtitre-plate based cytochrome P450 3A activity assay in fish cell lines. Environ. Toxicol. Chem. 2009, 28, 2632−2638. (33) Celander, M.; Hahn, M. E.; Stegeman, J. J. Cytochromes P450 (CYP) in the Poeciliopsis lucida hepatocellular carcinoma cell line (PLHC-1): Dose- and time-dependent glucocorticoid potentiation of CYP1A induction without induction of CYP3A. Arch. Biochem. Biophys. 1996, 329 (1), 113−22. (34) Baldwin, W. S.; Roling, J. A.; Peterson, S.; Chapman, L. M. Effects of nonylphenol on hepatic testosterone metabolism and the expression of acute phase’ proteins in winter flounder (Pleuronectes americanus): Comparison to the effects of Saint John’s Wort. Comp. Biochem. Physiol. C 2005, 140, 87−96. (35) Hahn, M. E.; Merson, R. R.; Karchner, S. I., Xenobiotic receptors in fish: Structural and functional diversity and evolutionary insights. In Environmental Toxicology; Moon, T. W., Mommsen, T. P., Eds. ;Elsevier, 2005; pp 191−228. (36) Aparicio, S.; Chapman, J.; Stupka, E.; Putnam, N.; Chia, J.; Dehal, P.; Christoffels, A.; Rash, S.; Hoon, S.; Smit, A.; Gelpke, M. D. S.; Roach, J.; Oh, T.; Ho, I. Y.; Wong, M.; Detter, C.; Verhoef, F.; Predki, P.; Tay, A.; Lucas, S.; Richardson, P.; Smith, S. F.; Clark, M. S.; Edwards, Y. J. K.; Doggett, N.; Zharkikh, A.; Tavtigian, S. V.; Pruss, D.; Barnstead, M.; Evans, C.; Baden, H.; Powell, J.; Glusman, G.; Rowen, L.; Hood, L.; Tan, Y. H.; Elgar, G.; Hawkins, T.; Venkatesh, B.; Rokhsar, D.; Brenner, S. Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 2002, 297, 1301− 1310. (37) Gerbal-Chaloin, S.; Pascussi, J. M.; Pichard-Garcia, L.; Daujat, M.; Waechter, F.; Fabre, J. M.; Carrere, N.; Maurel, P. Induction of CYP2C genes in human hepatocytes in primary culture. Drug Metab. Dispos. 2001, 29, 242−251. (38) Hasselberg, L.; Grøsvik, B. E.; Goksøyr, A.; Celander, M. C. Interactions between xenoestrogens and ketoconazole on hepatic CYP1A and CYP3A, in juvenile Atlantic cod (Gadus morhua). Comp. Hepatol. 2005, 4, 2. (39) Wang, H.; Huang, H.; Li, H.; Teotico, D. G.; Sinz, M.; Baker, S. D.; Staudinger, J.; Kalpana, G.; Redinbo, M. R.; Mani, S. Activated pregnenolone X-receptor is a target for ketoconazole and its analogs. Clin. Cancer Res. 2007, 13, 2488−2495. (40) Hegelund, T.; Ottosson, K.; Radinger, M.; Tomberg, P.; Celander, M. C. Effects of the antifungal imidazole ketoconazole on CYPIA and CYP3A in rainbow trout and killifish. Environ. Toxicol. Chem. 2004, 23, 1326−1334. (41) Hasselberg, L.; Westerberg, S.; Wassmur, B.; Celander, M. C. Ketoconazole, an antifungal imidazole, increases the sensitivity of rainbow trout to 17 alpha-ethynylestradiol exposure. Aquat. Toxicol. 2008, 86, 256−264. (42) Rae, J. M.; Johnson, M. D.; Lippman, M. E.; Flockhart, D. A. Rifampin is a selective, pleiotropic inducer of drug metabolism genes in human hepatocytes: Studies with cDNA and oligonucleotide expression arrays. J. Pharmacol. Exp. Ther. 2001, 299, 849−857. (43) Hayes, J. D.; Pulford, D. J. The glutathione S-transferase supergene family: Regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 1995, 30 (6), 445−600.

(44) Maglich, J. M.; Stoltz, C. M.; Goodwin, B.; Hawkins-Brown, D.; Moore, J. T.; Kliewer, S. A. Nuclear pregnane X receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. Mol. Pharmacol. 2002, 62, 638−646. (45) Celander, M.; Leaver, M. J.; George, S. G.; Förlin, L. Induction of cytochrome P4501A1 and conjugating enzymes in rainbow trout (Oncorhynchus mykiss) liver: A time course study. Comp. Biochem. Physiol.C 1993, 106 (2), 343−349. (46) George, S. G. Enzymology and molecular biology of phase II xenobiotic-conjugating enzymes in fish. In Aquatic Toxicology: Molecular, Biochemical and Cellular Perspectives; Ostrander, G. K., Malins, D. C., Eds.; CRC Press: Boca Raton, FL, 1994; pp 37−85. (47) Leaver, M. J.; George, S. G. Three repeated glutathione Stransferase genes from a marine fish, the plaice (Pleuronectes platessa). Mar. Environ. Res. 1996, 42, 19−23. (48) Henson, K. L.; Stauffer, G.; Gallagher, E. P. Induction of glutathione S-transferase activity and protein expression in brown bullhead (Ameiurus nebulosus) liver by ethoxyquin. Toxicol. Sci. 2001, 62, 54−60. (49) Burk, O.; Arnold, K. A.; Geick, A.; Tegude, H.; Eichelbaum, M. A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression. Biol. Chem. 2005, 386, 503−513. (50) Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug resistance in cancer: Role of ATP-dependent transporters. Nat. Rev. Cancer 2002, 2, 48−58. (51) Geick, A.; Eichelbaum, M.; Burk, O. Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J. Biol. Chem. 2001, 276, 14581−14587. (52) Mills, J. B.; Rose, K. A.; Sadagopan, N.; Sahi, J.; de Morais, S. M. F. Induction of drug metabolism enzymes and MDR1 using a novel human hepatocyte cell line. J. Pharmacol. Exp. Ther. 2004, 309, 303− 309. (53) Nishimura, M.; Koeda, A.; Suzuki, E.; Kawano, Y.; Nakayama, M.; Satoh, T.; Narimatsu, S.; Naito, S. Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes. Drug Metab. Pharmacok. 2006, 21, 297−307. (54) Dussault, I.; Lin, M.; Hollister, K.; Wang, E. H.; Synold, T. W.; Forman, B. M. Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR. J. Biol. Chem. 2001, 276, 33309−33312. (55) Kauffmann, H. M.; Pfannschmidt, S.; Zoller, H.; Benz, A.; Vorderstemann, B.; Webster, J. I.; Schrenk, D. Influence of redoxactive compounds and PXR-activators on human MRP1 and MRP2 gene expression. Toxicology 2002, 171, 137−146. (56) Kauffmann, H. M.; Keppler, D.; Gant, T. W.; Schrenk, D. Induction of hepatic mrp2 (cmrp/cmoat) gene expression in nonhuman primates treated with rifampicin or tamoxifen. Arch. Toxicol. 1998, 72, 763−768. (57) Schrenk, D.; Baus, P. R.; Ermel, N.; Klein, C.; Vorderstemann, B.; Kauffmann, H. M. Up-regulation of transporters of the MRP family by drugs and toxins. Toxicol. Lett. 2001, 120, 51−57. (58) Bard, S. M. Multixenobiotic resistance as a cellular defense mechanism in aquatic organisms. Aquat. Toxicol. 2000, 48, 357−389. (59) Long, Y.; Li, Q.; Zhong, S.; Wang, Y.; Cui, Z. Molecular characterization and functions of zebrafish ABCC2 in cellular efflux of heavy metals. Comp. Biochem. Physiol. C 2011, 153, 381−391. (60) Caminada, D.; Zaja, R.; Smital, T.; Fent, K. Human pharmaceuticals modulate P-gp1 (ABCB1) transport activity in the fish cell line PLHC-1. Aquat. Toxicol. 2008, 90, 214−222. (61) Annilo, T.; Chen, Z. Q.; Shulenin, S.; Costantino, J.; Thomas, L.; Lou, H.; Stefanov, S.; Dean, M. Evolution of the vertebrate ABC gene family: Analysis of gene birth and death. Genomics 2006, 88, 1− 11. (62) Bresolin, T.; Rebelo, M. D.; Bainy, A. C. D. Expression of PXR, CYP3A and MDR1 genes in liver of zebrafish. Comp. Biochem. Physiol. C 2005, 140, 403−407. (63) Svecova, L.; Vrzal, R.; Burysek, L.; Anzenbacherova, E.; Cerveny, L.; Grim, J.; Trejtnar, F.; Kunes, J.; Pour, M.; Staud, F.; 6313

dx.doi.org/10.1021/es3005305 | Environ. Sci. Technol. 2012, 46, 6306−6314

Environmental Science & Technology

Article

Anzenbacher, P.; Dvorak, Z.; Pavek, P. Azole antimycotics differentially affect rifampicin-induced pregnane x receptor-mediated CYP3A4 gene expression. Drug Metab. Dispos. 2008, 36, 339−348. (64) Courtois, A.; Payen, L.; Guillouzo, A.; Fardel, O. Up-regulation of multidrug resistance-associated protein 2 (MRP2) expression in rat hepatocytes by dexamethasone. FEBS Lett. 1999, 459, 381−385. (65) Vaccaro, E.; Salvetti, A.; Del Carratore, R.; Nencioni, S.; Longo, V.; Gervasi, P. G. Cloning, tissue expression, and inducibility of CYP 3A79 from sea bass (Dicentrarchus labrax). J. Biochem. Mol. Toxic. 2007, 21, 32−40. (66) Aouabdi, S.; Gibson, G.; Plant, N. Transcriptional regulation of the PXR gene: Identification and characterization of a functional peroxisome proliferator-activated receptor alpha binding site within the proximal promoter of PXR. Drug Metab. Dispos. 2006, 34, 138− 144. (67) Zhang, H.; LeCulyse, E.; Liu, L.; Hu, M. W.; Matoney, L.; Zhu, W. Z.; Yan, B. F. Rat pregnane X receptor: Molecular cloning, tissue distribution, and xenobiotic regulation. Arch. Biochem. Biophys. 1999, 368, 14−22. (68) Shaban, Z.; Soliman, M.; El-Shazly, S.; El-Bohi, K.; Abdelazeez, A.; Kehelo, K.; Kim, H. S.; Muzandu, K.; Ishizuka, M.; Kazusaka, A.; Fujita, S. AhR and PPAR alpha: Antagonistic effects on CYP2B and CYP3A, and additive inhibitory effects on CYP2C11. Xenobiotica 2005, 35, 51−68. (69) Cui, X.; Thomas, A.; Gerlach, V.; White, R. E.; Morrison, R. A.; Cheng, K. C. Application and interpretation of hPXR screening data: Validation of reporter signal requirements for prediction of clinically relevant CYP3A4 inducers. Biochem. Pharmacol. 2008, 76, 680−689. (70) Thibaut, R.; Schnell, S.; Porte, C. The interference of pharmaceuticals with endogenous and xenobiotic metabolizing enzymes in carp liver: An in-vitro study. Environ. Sci. Technol. 2006, 40, 5154−5160. (71) vanLieshout, E. M. M.; Tiemessen, D. M.; Peters, W. H. M.; Jansen, J. Effects of nonsteroidal anti-inflammatory drugs on glutathione S-transferases of the rat digestive tract. Carcinogenesis 1997, 18, 485−490. (72) Lehmann, J. M.; Lenhard, J. M.; Oliver, B. B.; Ringold, G. M.; Kliewer, S. A. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal antiinflammatory drugs. J. Biol. Chem. 1997, 272, 3406−3410. (73) Sinz, M.; Kim, S.; Zhu, Z.; Chen, T.; Anthony, M.; Dickinson, K.; Rodrigues, A. D. Evaluation of 170 xenobiotics as transactivators of human pregnane X receptor (hPXR) and correlation to known CYP3A4 drug interactions. Curr. Drug Metab. 2006, 7, 375−388.

■

NOTE ADDED AFTER ASAP PUBLICATION Minor changes to the figures and throughout the text were made to the version of this paper published May 10, 2012. The correct version published May 11, 2012.

6314

dx.doi.org/10.1021/es3005305 | Environ. Sci. Technol. 2012, 46, 6306−6314