ToxSci Advance Access originally published online on March 10, 2008
Toxicological Sciences 2008 103(2):268-277; doi:10.1093/toxsci/kfn047
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Phthalate Induction of CYP3A4 is Dependent on Glucocorticoid Regulation of PXR Expression
Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695
1 To whom correspondence should be addressed at Department of Environmental and Molecular Toxicology, Box 7633, North Carolina State University, Raleigh, NC 27695-7633. Fax: (919) 515-7169. E-mail: andrew_wallace{at}ncsu.edu.
Received December 24, 2007; accepted February 28, 2008
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Cytochrome P450 3A4 (CYP3A4) is responsible for oxidative metabolism of more than 60% of all pharmaceuticals. CYP3A4 is inducible by xenobiotics that activate pregnane X receptor (PXR), and enhanced CYP3A4 activity has been implicated in adverse drug interactions. Recent evidence suggest that the widely used plasticizer, di-2-ethylhexyl phthalate (DEHP), and its primary metabolite mono-2-ethylhexyl phthalate (MEHP) may act as agonists for PXR. Hospital patients are uniquely exposed to high levels of DEHP as well as being administered glucocorticoids. Glucocorticoids positively regulate PXR expression in a glucocorticoid receptor (GR)–mediated mechanism. We suggest that the magnitude of CYP3A4 induction by phthalates is dependent on the expression of PXR and may be significantly higher in the presence of glucocorticoids. DEHP and MEHP induced PXR-mediated transcription of the CYP3A4 promoter in a dose-dependent fashion. Coexposure to phthalates and dexamethasone (Dex) resulted in enhanced CYP3A4 promoter activity; furthermore, this induction was abrogated by both the GR antagonist RU486 and GR small interfering ribonucleic acid. Dex induced PXR protein expression in human hepatocytes and a liver-derived rat cell line. CYP3A4 protein was highly induced by Dex and DEHP coadministration in human hepatocyte cultures. Finally, enhanced 6β-hydroxytestosterone formation in Dex and phthalate cotreated human hepatocytes confirmed CYP3A4 enzyme induction. Concomitant exposure to glucocorticoids and phthalates resulting in enhanced metabolic activity of CYP3A4 may play a role in altered efficacy of pharmaceutical agents. Understanding the role of glucocorticoid regulation of PXR as a key determinant in the magnitude of CYP3A4 induction by xenobiotics may provide insight into adverse drug effects in a sensitive population.
Key Words: pregnane X receptor; CYP3A4; DEHP; MEHP; glucocorticoid.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Di-2-ethylhexyl phthalate (DEHP) is a widely used plasticizer for medical products such as IV fluid bags, blood bags and tubing. Phthalate plasticizers are not chemically bound to the plastic matrix and leach out of polyvinyl chloride products or are extracted from plastics by lipophilic drug formulations (Tickner et al., 2001
). Latini et al., detected mean serum concentrations of 1.19 µg/ml (3.0µM) DEHP and 0.52 µg/ml (1.9µM) mono-2-ethylhexyl phthalate (MEHP) in the cord blood of newborns, suggesting that exposure can begin in utero (Latini et al., 2004). The levels of DEHP exposure to the general population range from 3 to 30 µg/kg/day and blood levels of DEHP and MEHP are approximately 5.7 ng/ml (15nM) (Kavlock et al., 2006). Hospital patients undergoing extracorporeal membrane oxygenation (ECMO), mechanical ventilation, total parenteral nutrition (TPN), and multiple transfusions could be uniquely exposed in these extreme cases to high DEHP levels resulting in exposures between 0.005 and 8.5 mg/kg bw/day (FDA 2004; Kavlock et al., 2006; Kim et al., 2005; Koch et al., 2006; Loff et al., 2000). Concern has been raised due to reports indicating that hospitalized neonates receiving intensive therapeutic interventions are exposed to DEHP levels up to two to three orders of magnitude above exposure for the general population (Frederiksen et al., 2007; Shea, 2003). Following a single exchange transfusion with catheters, neonate DEHP serum levels were found to range from 6.1 to 21.6 µg/ml (1.9–6.0µM) (Plonait et al., 1993). Research has linked DEHP to a variety of adverse effects including hepatotoxicity, nephrotoxicity, teratogenicity, and testicular toxicity (Shiota and Nishimura, 1982; Ward et al., 1998). The developing animal is susceptible to the endocrine disrupting effects of phthalates, and studies have shown that exposure to high levels of DEHP causes testicular dysgenesis syndrome in rodents (Foster et al., 2001; Tickner et al., 2001). The National Toxicology Program Center for the Evaluation of Risks to Human Reproduction (NTP-CERHR) recently released an evaluation of the scientific evidence that DEHP is a reproductive or developmental toxicant, and indicated the risk to newborn infants undergoing multiple medical procedures is greatest, as they are exposed to especially high levels (Kavlock et al., 2006).
It is well established that the peroxisome proliferative and hepatocarcinogenic effects of phthalates are mediated by the peroxisome proliferator–activated receptor (PPAR-
). Research showing that DEHP-induced renal and testicular toxicities are mediated in a PPAR-–independent fashion suggests that other signaling pathways play a role in eliciting the toxicological effects of phthalates (Ward et al., 1998). Hurst and Waxman (2004) showed that the pregnane X receptor (PXR) is activated by MEHP and other phthalates. PXR is considered to be a master regulator of xenobiotic metabolism, capable of binding response elements in the promoter regions of many metabolic genes.
PXR, a member of the nuclear receptor superfamily, has a unique ligand binding pocket able to bind a variety of diverse compounds known to induce target gene expression (Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer et al., 1998, 2002). With the identification of PXR, a novel signaling pathway for CYP3A induction by xenobiotics was discovered. Cytochrome P450 3A4 (CYP3A4), the most abundant Phase I enzyme in human liver, is responsible for the oxidative metabolism of more than 60% of pharmaceuticals (Li et al., 1995). CYP3A4 gene expression is inducible by numerous xenobiotics, resulting in altered drug metabolism and drug–drug interactions in addition to enhanced metabolism of endogenous substrates including testosterone. Xenobiotic response elements in the proximal promoter and distal enhancer regions of the CYP3A4 gene have been identified as PXR binding sites.
Glucocorticoids are steroid hormones, essential for normal growth and development, liver and immune functions, and in mediating stress responses. The actions of glucocorticoids are mediated by binding to the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. Physiological circulating levels of the endogenous glucocorticoid cortisol range from 0.1 to 0.45µM (Brien, 1980). Dexamethasone (Dex), a widely prescribed synthetic glucocorticoid, is used in the treatment of cancer and immune disorders, and during perinatal, neonatal, and postnatal periods to aid lung development and to treat or prevent chronic lung disease due to mechanical ventilation or ECMO (Griffin et al., 2004; Halliday, 2004; Heggen et al., 2004; Raff, 2004; Thebaud et al., 2001). Therapeutic blood levels of Dex are in the nanomolar range and rarely reach micromolar concentrations (Lehmann et al., 1998). Nanomolar concentrations of glucocorticoids, such as Dex, play a critical role in CYP3A4 expression and induction through a process involving GR-mediated PXR messenger RNA (mRNA) accumulation (Pascussi et al., 2000). These findings led to a proposed two-stage model of CYP3A induction involving GR-mediated induction of PXR expression by glucocorticoids and subsequent PXR-mediated induction of CYP3A by PXR ligands (Huss and Kasper, 2000; Pascussi et al., 2001). Based on this model, we suggest that the magnitude of CYP3A4 induction by phthalates is dependent on the expression of PXR and may be significantly higher in the presence of glucocorticoids. No studies to date have simultaneously investigated the effects of the concomitant exposure to glucocorticoids and phthalates in humans. Patients being treated with glucocorticoids for cancer, to lessen the effects of mechanical ventilation or ECMO (Halliday, 2004; Raff, 2004; Rhen and Cidlowski, 2005; Thebaud et al., 2001; Wallace and Cidlowski, 2002) are also exposed to high levels of DEHP as it leaches out of or is extracted from polyvinyl chloride medical devices (Bagel-Boithias et al., 2005; Griffin et al., 2004; Heggen et al., 2004; Kim et al., 2005; Tickner et al., 2001). Coexposure to glucocorticoids and phthalates resulting in enhanced metabolic activity of CYP3A may play a role in accelerating the metabolism of endogenous CYP3A4 substrates and alter the efficacy of pharmaceutical agents that are CYP3A4 substrates.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Chemicals.
1,4-Pregnadien-9-fluoro-16 -methyl-11, 17, 21-triol-3,20-dione (Dex), 4-androsten-17β-ol-3-one (testosterone), and 4-androsten-6β,17β-diol-3-one (6β-OHTST) were purchased from Steraloids, Inc. (Newport, RI). Rifampicin (Rif), Mifepristone (RU486), DEHP and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St Louis, MO). MEHP was purchased from TCI America (Portland, OR). All cell culture media and supplements were purchased from Mediatech, Inc. (Herndon, VA) unless otherwise noted. All primary hepatocyte culture medium and reagents were GIBCO brand, purchased from Invitrogen Corp. (Carlsbad, CA).
Plasmids and siRNA.
The plasmid CYP3A4-luc, a kind gift of Dr Jean-Marc Pascussi, contains –7836/–7208 nt of the distal enhancer region (XREM) cloned 5' of –263/+11 nt of the proximal promoter of the CYP3A4 gene cloned into the pGL3 reporter vector (Promega, Madison, WI). The plasmid PXR-pSG5, a kind gift of Dr Steven Kliewer, contains hPXR.1 cloned into the pSG5 expression vector (Stratagene, La Jolla, CA). Empty pSG5 vector was used as a control. The ON-TARGETplus siRNAs targeting rat GR (NR3C1, NM_012576
[GenBank]
) and control nontargeting siRNA #3 were purchased from Dharmacon (Lafayette, CO). The positive control, GRE2-luc plasmid, contains two glucocorticoid responsive elements (GREs) and is glucocorticoid responsive. The control vector pSV-Beta-galactosidase was purchased from Promega (Madison, WI).
Cell culture and transactivation experiments.
The human hepatocellular carcinoma, HepG2, cell line and the rat hepatoma H4IIE-C3 cell line were purchased from the American Type Culture Collection (ATCC, Manassas, VA), and cultured according to ATCC recommendations. HepG2 cells were transfected in Optimem-minimal essential medium (MEM) I reduced serum medium (Invitrogen Corp.) using TransIT-LT1 reagent (Mirus Bio Corp., Madison, WI) according to the manufacturer's recommendations. The cells were incubated with transfection reagent for 4–6 h, then the Opti-MEM medium was removed, and cells were rinsed once and incubated overnight in Cellgro Complete medium. For transfection of the H4IIE-C3 cell line, plated cells were washed with Opti-MEM and transfected using Lipofectamine reagent enhanced by Plus reagent (Invitrogen Corp.) according to the manufacturer's recommendations. The cells were incubated with transfection reagents for 3 h before being rinsed once and incubated overnight in complete medium supplemented with 10% dextran-charcoal stripped fetal bovine serum.
Analysis of luciferase activity.
Following transfection, cells were allowed to recover overnight in complete medium and then treated with vehicle or specified compound. After 48 h of treatment, cells were rinsed with phosphate buffered saline (PBS) and harvested in 1x Reporter Lysis Buffer from the Luciferase Assay System (Promega Corp.). Cell lysates were prepared according the manufacturer's recommended protocol and luciferase activity was measured using a TD-20/20 Luminometer by Turner Designs, Inc. (Sunnyvale, CA). As an internal transfection control, β-galactosidase assays were performed on cell lysates according to the recommended protocol in the β-Galactosidase Enzyme Assay System (Promega). Luciferase activity was normalized to either β-galactosidase activity or to total protein as indicated and then transformed to fold induction over control.
Human hepactocyte culture.
Freshly isolated human hepatocytes plated with Matrigel overlay were received from ADMET (RTP, NC), CellzDirect (Pittsboro, NC), or Cambrex (Walkersville, MD). In primary cultures of human hepatocytes, Matrigel overlay aids in maintaining differentiated characteristics of the cells as well as facilitating the induction of xenobiotic metabolizing enzymes (Gross-Steinmeyer et al., 2005). Cells were cultured for 2–3 days in William's E medium with insulin, transferrin, selenium A (Invitrogen, Calsbad, CA), 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and free of Dex. Medium was changed every 24 h during culture and treatment periods. For western blotting, hepatocytes seeded in 6 well plates were treated for 2 consecutive days and cellular lysates were prepared as described below. For testosterone hydroxylase assays, hepatocytes seeded in 24- or 48-well plates were treated for 3 consecutive days. LeCluyse et al. (2000) demonstrated that maximal activity of CYP3A4 was achieved after 3 days of exposure to inducing agents. The medium was aspirated 24 h following the final treatment and cells were rinsed two times and then incubated with 100µM testosterone substrate in fresh medium for 30 min. Medium was removed and combined with equal volume of methanol, vortexed briefly and centrifuged at 21,000 x g for 5 min. The supernatant was used for subsequent high-performance liquid chromatography (HPLC) analysis.
Analysis of 6β-OHTST by HPLC.
The generation of metabolites was analyzed using a Waters 2695 HPLC system equipped with a 2996 photodiode array detector (Waters, Milford, MA). The HPLC system was equipped with a degasser and an autoinjector and Waters Empower software ver. 5.0 was utilized for data collection and analysis. The mobile phase for pump A was 5% tetrahydrofuran, 95% water and for pump B was 100% methanol. The flow rate was 0.5 ml/min and a gradient system was employed as described previously (Usmani et al., 2003). Metabolites were separated on an Ultracarb column (Ultracarb 5 µm, 150 mm x 4.6 mm, octadecylsilica [30) (Phenomenex, Rancho Palos Verdes, CA) and detected at 240nM. 6β-OHTST was detected at the retention time of 15.8 min as determined by the use of a 6β-OHTST standard.
Western blot analysis.
For PXR detection, cellular lysates were prepared by resuspending and sonicating the cell pellet in NP-40 Lysis Buffer (25mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 10mM ethylene diamine tetraacetic acid (EDTA), 10mM ethylene glycol tetraacetic acid (EGTA) 3mM MgCl2, 10% glycerol, 20mM sodium molybdate and 0.5% IGEPAL® (octylphenyl-polyethylene glycol) containing protease inhibitor cocktail (Complete Mini, Roche Diagnostics, Indianapolis, IN). For CYP3A4 enzyme detection, S9 fractions were obtained after centrifugation of cell lysates at 19,000 x g. All samples were prepared in Laemmli Buffer containing β-mercaptoethanol and boiled for 5 min. Protein samples were resolved on 8% Novex Tris-Glycine gels (Invitrogen Corp.) and transferred to nitrocellulose membrane. Immunoreactive PXR was detected using an anti-hPXR epitope-specific polyclonal antibody (1:500) raised in rabbits (Covance) followed by detection with horseradish peroxidase–conjugated anti-rabbit secondary antibody. CYP3A4 was detected using a monoclonal mouse anti-CYP3A antibody (Gentest, BD Biosciences) (1:500) and a goat anti-mouse IRDye680 fluorescently labeled secondary antibody. β-Actin was detected using rabbit anti-β-actin primary antibody (Sigma) and either a horseradish peroxidase–conjugated anti-rabbit secondary antibody or goat anti-rabbit IRDye800 fluorescently labeled secondary antibody. All primary antibodies were incubated overnight at 4°C in tris buffered saline (TBS) or phosphate buffered saline (PBS) with Tween and 1% nonfat dry milk. Immunoblots were visualized using chemiluminescence detection (Enhanced chemiluminescence, GE Healthcare Biosciences (Piscataway, NJ)) or with a LiCOR Odyssey Infrared Imaging System. Densitometric analysis of immunoreactive protein bands was performed using a Kodak Image Station 440 CF with Kodak Molecular Imaging Software (Rochester, NY). The integrated intensity of CYP3A4 protein and β-actin levels was determined by LiCOR Odyssey Imaging Software (Lincoln, NE).
Statistical analysis.
All statistical analysis was performed using JMP software, version 6.0.0 (SAS Institute, Inc., Cary, NC). Analysis was done using ANOVA followed by comparisons of the treatment means with control (untreated) using Dunnett's method with a significance level of 0.05. Comparisons between all treatment and control groups were made using ANOVA followed by Tukey–Kramer Honestly Significant Difference (HSD) with a significance level of 0.05. Treatments not connected by the same letter were found to be significantly different. 6β-OHTST levels were log-transformed to eliminate heteroscedasticity between individual human hepatocyte donors as described by Rhen et al. (1999). Transformed 6β-OHTST levels were also analyzed using ANOVA followed by Tukey–Kramer HSD with a significance level of 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
It is well established that PXR-mediated induction of CYP3A4 can result in altered metabolism of pharmaceuticals and endogenous steroids, which may lead to adverse drug interactions. The ability of glucocorticoids to positively regulate PXR expression may be a key determinant in the magnitude of CYP3A4 induction by xenobiotics, such as the phthalate esters DEHP and MEHP. To investigate the ability of DEHP to activate human PXR and induce CYP3A4 promoter activity, HepG2 cells were transiently transfected with expression plasmid containing PXR or empty vector and luciferase reporter containing a portion of the CYP3A4 promoter with three PXR binding sites (Fig. 1A). In the presence of PXR, 10µM DEHP induced CYP3A4 promoter activity 22-fold above that of vehicle control, whereas Rif, the prototypical ligand for hPXR (Zhang et al., 1999
), induced the CYP3A4 promoter 28-fold over control. The CYP3A4 promoter was not activated in the absence of PXR for any of the treatment groups.
|
DEHP is rapidly metabolized after all routes of uptake; the first step being cleavage of DEHP into the monoester MEHP, which is then further metabolized by various oxidation reactions (Albro and Thomas, 1973
; Schmid and Schlatter, 1985). We investigated the ability of the primary metabolite, MEHP, to activate PXR and induce CYP3A4 promoter activity. HepG2 cells were utilized in transient transfection studies with the expression plasmid for PXR and CYP3A4-luciferase reporter. Both DEHP and MEHP induced CYP3A4 promoter activity in a dose-dependent manner in the presence of PXR, with maximal activation at 25µM for both compounds (Fig. 1B). Neither DEHP nor MEHP activated the promoter construct in the absence of PXR (data not shown). At each concentration, DEHP induction of CYP3A4 was greater than MEHP. Patients undergoing multiple medical procedures are uniquely exposed to high levels of phthalates and many may also be administered glucocorticoids, such as Dex. We evaluated the effects of coexposure to Dex and DEHP or MEHP on CYP3A4 promoter activity. Transient transfection of rat hepatoma H4IIE-C3 cells was performed using the CYP3A4-luciferase reporter. H4IIE-C3 cells are glucocorticoid responsive and express PXR. The CYP3A4-luciferase reporter was induced approximately 10-fold by 10µM DEHP and 5-fold by 0.1µM Dex, but cotreatment with Dex and DEHP dramatically induced CYP3A4 activity 65-fold over vehicle treated control (Fig. 2A). Coexposure to 0.1µM Dex and 10µM MEHP resulted in 12-fold induction of CYP3A4 promoter activity over vehicle treated control (Fig. 2B). This led us to hypothesize that GR plays a critical role in the induction of CYP3A4 by glucocorticoids and phthalates. To further elucidate the role of GR in this process, transfected H4IIE-C3 cells were treated with the glucocorticoid antagonist RU486. When 0.1µM RU486 was administered in addition to Dex and DEHP, CYP3A4 promoter activity was reduced more than 50% to approximately 30-fold over vehicle treated control (Fig. 3A). Additionally, when 0.1µM RU486 was administered in conjunction with Dex and MEHP, CYP3A4-luciferase reporter activity was reduced to 5-fold over control; less than half of the induction seen with Dex and MEHP coadministration (Fig. 3B). The GR antagonist RU486 effectively abrogates the coordinate Dex and phthalate induction of CYP3A4 promoter activity.
|
|
To further examine the role of GR, H4IIE-C3 cells were transfected with either rat GR-targeting siRNAs or control siRNAs. When Dex and DEHP were coadministered in the presence of control siRNAs, CYP3A4 promoter activity was induced. In cells transfected with rat GR siRNAs, CYP3A4-luciferase reporter activity was reduced to 3.4-fold over the control; less than 25% of the induction seen by concomitant Dex and DEHP administration (Fig. 4A). As an additional control, GRE2-Luc and siRNAs were transfected into H4IIE-C3 cells. The control siRNAs had no effect on glucocorticoid responsiveness, but siRNAs targeting rat GR abrogated Dex induction of GRE2-Luc activity (Fig. 4B). The critical role of GR-dependent PXR expression in the DEHP induction of CYP3A4-Luc was investigated by transiently cotransfecting GR-targeting siRNA and null expression plasmid or a PXR expressing plasmid. No significant induction of CYP3A4 promoter activity was observed by treatments in the presence of the null expression vector and GR siRNA. In contrast, CYP3A4 promoter activity was significantly induced over 100-fold in cells transfected with PXR and treated with DEHP or DEHP and Dex, with no significant difference being observed between treatments containing DEHP (Fig. 4C).
|
It's been shown that nanomolar concentrations of Dex increase PXR mRNA expression (Pascussi et al., 2000
) and to evaluate the ability of low-dose Dex to induce PXR protein expression we utilized H4IIE-C3 cells and freshly isolated human hepatocytes. Western blot analysis was performed on cellular lysates prepared after 48 h of treatment with increasing doses of Dex (Figs. 5A and 5B). In H4IIE-C3 rat hepatoma cells, rPXR protein expression was enhanced by 0.01 and 0.1µM Dex in a dose-dependent manner, over that of basal expression (Fig. 5A). Human PXR protein was induced over control in all Dex treated samples in a dose-dependent manner (Fig. 5B).
|
After observing that Dex and DEHP coordinately induced CYP3A4 promoter activity in transactivation assays, we were interested in evaluating the effect of cotreatment with Dex and DEHP on CYP3A4 protein expression. Freshly isolated human hepatocyte cultures were treated with DEHP in the presence or absence of Dex and Western blot analysis was performed on cellular lysates to visualize the levels of CYP3A4 protein (Fig. 6A). As indicated by densitometric analysis, treatment with Dex alone induced CYP3A4 protein approximately 2-fold over control, whereas DEHP alone induced CYP3A4 protein levels 3-fold, but was not statistically significant (Fig. 6B). However, in the presence of Dex, DEHP induced CYP3A4 protein to 13-fold over basal expression level.
|
To assess if changes observed in CYP3A4 protein expression resulted in altered CYP3A4 enzymatic activity, testosterone was used as a specific substrate probe. The utility of measuring testosterone 6β-hydroxylase activity in intact human hepatocyte cultures to monitor CYP3A4 activity has been demonstrated previously (Fayer et al., 2001
; Kostrubsky et al., 1999). Formation of the 6β-OHTST metabolite can be correlated to CYP3A4 enzymatic activity (LeCluyse et al., 2000; Waxman et al., 1988). CYP3A4 activity was strongly induced by 10µM Rif, a known inducer of CYP3A4 (Fig. 7). This effect was greatly enhanced by the presence of Dex. DEHP and MEHP had no effect on CYP3A4 activity when administered in the absence of Dex (Fig. 7). However, coadministration of Dex resulted in a phthalate dose-dependent increase in 6β-OHTST formation.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
We have investigated the effects of concomitant exposure to Dex and the phthalate esters, DEHP and MEHP, on CYP3A4 expression and activity. Enhanced CYP3A4 activity has been implicated in cases of drug–drug interactions, decreased efficacy of pharmaceutical agents, and increased toxicity via bioactivation of therapeutics. DEHP and MEHP fall in the broad category of peroxisome proliferators, exhibiting many of their hepatotoxic effects through activation of the PPAR-
(Maloney and Waxman, 1999; Ward et al., 1998). PPAR- is a member of the nuclear receptor family and is expressed in numerous tissues important in lipid metabolism (Braissant et al., 1996). Studies have shown that exposure to peroxisome proliferators leads to alterations in expression of steroidogenic enzymes involved in the conversion of cholesterol to testosterone (Lehmann et al., 2004) as well as increased expression of cytochrome P450s responsible for metabolism of testosterone, including members of the CYP2C and CYP3A families (Kim et al., 2003). However, evidence that PPAR- null mice develop renal and testicular toxicity in response to DEHP exposure (Ward et al., 1998) presents the possibility that phthalates may elicit toxicity through additional signaling pathways involving other nuclear receptors.
Similar to PPAR-, PXR is a member of the nuclear receptor superfamily. It is involved in regulation of metabolic genes and is expressed in liver, kidney, and intestinal tissues. PXR binds a variety of response elements in promoters of genes involved in transport, metabolism and elimination of bile acids, steroids and xenobiotics; most notably, CYP3A4 (Kliewer et al., 2002). The most abundant P450 in adult human liver, CYP3A4 plays a pivotal role in drug metabolism and maintenance of endogenous steroid hormone levels. PXR responsive regions within the CYP3A gene promoter confer high inducibility by xenobiotics (Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer et al., 1998). Such induction leads to accelerated metabolism of pharmaceuticals and altered pharmacokinetic and pharmacodynamic properties, as well as enhanced testosterone metabolism. Findings in rodent models have shown DEHP is able to induce CYP3A in testes and liver resulting in increased testosterone metabolism (Kim et al., 2003; Masuyama et al., 2000).
The results of our initial HepG2 cell reporter assays demonstrated that DEHP, as well as the monoester metabolite MEHP, activated PXR-mediated transcription of the CYP3A4 target gene. The ability of MEHP and other phthalate monoesters to activate mPXR and hPXR was demonstrated previously (Hurst and Waxman, 2004), but the ability of the parent compound DEHP to act as a PXR ligand has not been well studied. We have shown here that DEHP is actually more efficacious than MEHP at activating hPXR, reaching maximal induction of the CYP3A4-luciferase reporter at 25µM. In human colon carcinoma cells, DEHP was shown to activate PXR-mediated transcription of the MDR1 gene promoter. Additionally, the LBD of human PXR associated with the receptor interacting domain of the coactivator SRC-1 in the presence of 10µM DEHP in mammalian two-hybrid assays (Takeshita et al., 2006). Taken together, these data suggest that DEHP and MEHP may be ligands for PXR and initiate PXR-mediated transcription of target genes. Alternatively, DEHP and MEHP may cause the release of an endogenous PXR ligand.
Historically, CYP3A family members across species have demonstrated responsiveness to glucocorticoids, but the Dex responsive sites in CYP3A promoters do not contain GREs, and the GR does not associate with these elements (Huss et al., 1996; Quattrochi et al., 1995). Additionally, the time course of CYP3A induction following glucocorticoid treatment does not follow that of other GR-regulated genes, suggesting that the regulation of CYP3A induction is a secondary effect of GR activation (Schuetz and Guzelian, 1984). Pascussi et al. (2000) demonstrated that at supraphysiological micromolar concentrations, Dex can act as a ligand for human PXR and subsequently induce CYP3A4, whereas treatment with the antiglucocorticoid, RU486, was shown to inhibit CYP3A4 protein expression. Pretreatment of human hepatocytes with the translational inhibitor cycloheximide was shown to inhibit PXR-mediated CYP3A4 gene expression resulting from treatment with submicromolar concentrations of Dex, but had no effect on PXR mRNA accumulation (Pascussi et al., 2000). These findings led to a proposed two-stage model of CYP3A induction, explaining the secondary effect of GR activation, by GR-mediated induction of PXR expression by nanomolar concentrations of Dex and subsequent PXR-mediated induction of CYP3A by PXR ligands (Huss and Kasper, 2000; Pascussi et al., 2001).
We have demonstrated here that CYP3A4 promoter activity is significantly increased when DEHP and MEHP are administered in the presence of Dex. This induction of CYP3A4 transcription is strongly abrogated by coadministration of the GR antagonist, RU486, and by transfection with rat GR-targeting siRNA, further confirming that GR activation is necessary for synergistic CYP3A4 induction by glucocorticoid and PXR agonist coexposure. Due to low transfection efficiency of the H4IIE-C3 cell line, Western blotting of rat GR-targeting siRNA transfected H4IIE-C3 cell lysates failed to measure any significant reduction in GR or PXR levels (data not shown). To further demonstrate that PXR expression is critical for DEHP-induced CYP3A4 promoter activity, we transiently transfected GR-targeting siRNA with a PXR expression plasmid. In cells transiently transfected with PXR, DEHP induction of CYP3A4 promoter activity was rescued and no significant differences were found between cells treated with DEHP alone or coadministered DEHP and Dex. These findings suggest that the increase in PXR expression in response to DEHP and Dex is mediated by GR, and not by other confounding effects of Dex. The H4IIE-C3 rat hepatoma cell line utilized in these reporter assays is an excellent model for evaluating the combined effects of GR and PXR activation. These cells posses a functional GR as well as PXR (Huss and Kasper, 1998), so we were able to measure the effects of our treatments on the endogenous receptors and minimize the artificial nature of a cell-based reporter assay. Huss and Kasper (2000) demonstrated synergistic induction of the rat CYP3A23 promoter in H4IIE cells by cotreatment with 10µM pregnenolone-16-carbonitrile, an agonist for rat PXR, and 0.1µM Dex. The resulting transcriptional activity far exceeded that reached with either individual treatment, and our current data agree nicely with their previous findings.
Nanomolar concentrations of Dex increase PXR mRNA expression in human hepatocytes via enhanced transcription of PXR (Pascussi et al., 2000). We have shown that similar concentrations of Dex induced rat and human PXR protein expression in the H4IIE-C3 cell line and freshly isolated human hepatocytes. In both cases, a dose-dependent increase in PXR protein was observed. Evidence that GR positively regulates the expression of PXR may account for the synergistic effect of Dex and PXR activators on CYP3A induction. In cultured human hepatocytes, concomitant addition of Dex enhanced CYP3A4 mRNA induction by Rif with maximal induction at 0.1µM Dex, a concentration known to fully activate the GR (Pascussi et al., 2000). Additionally, we demonstrated that human hepatocyte cultures coexposed to DEHP and Dex have highly induced CYP3A4 protein levels when compared to basal CYP3A4 expression or to resulting CYP3A4 levels from either individual treatment. We hypothesize that due to low basal PXR expression in human hepatocytes, endogenous CYP3A4 levels are low, and that GR stimulation by submicromolar levels of glucocorticoids, such as Dex, is needed to increase PXR expression in order to see any effect on CYP3A4 by PXR agonists. The addition of Dex and activation of GR may also recruit necessary coactivators to the transcriptional assembly, thus promoting PXR stimulation of CYP3A4 transcription.
The significance of enhanced CYP3A4 protein expression can be demonstrated by measuring functional increases in testosterone hydroxylase activity and formation of the 6β-OHTST metabolite in human hepatocyte cultures. We have shown here that DEHP and MEHP, as well as Rif, dramatically enhanced 6β-OHTST production when coadministered with Dex in human hepatocytes. Our finding that both phthalates did not induce testosterone hydroxylase activity when given in the absence of Dex was expected, because DEHP did not significantly induce CYP3A4 protein levels when given alone.
DEHP is a ubiquitous environmental contaminant resulting in daily ambient exposure for the general population, but various subpopulations are exposed to extremely high levels of this phthalate ester and its primary metabolite MEHP. The latest NTP-CERHR report indicated that DEHP exposure to the general population is between 3 and 30 µg/kg/day and is of minimal concern (Kavlock et al., 2006). Additionally, estimates of exposure based on the levels of urinary MEHP oxidative metabolites, recently recognized as more accurate measures of DEHP exposure, indicate that the highest exposures in the general population (95th percentile) were 65 µg/kg bw/day for men and 27.4 µg/kg bw/day for women. (Kavlock et al., 2006; Koch et al., 2005, 2006). Especially high DEHP exposure to patients occurs due to ECMO, mechanical ventilation, total parenteral nutrition, extraction by anticancer drug formulations, and multiple transfusions and results in DEHP exposure levels between 0.005 and 8.5 mg/kg bw/day (FDA 2004; Kavlock et al., 2006; Kim et al., 2005; Koch et al., 2006; Loff et al., 2000). In a study using deuterium labeled DEHP administered to a human volunteer at a dose of 650 µg/kg body weight, MEHP serum levels were measured at 4.95 mg/l (17.8µM) two hours after exposure and within 8 h fell to 0.36µM, which is within the range of MEHP used in our studies (Koch et al., 2004, 2005). Although further investigation is needed to determine the levels of DEHP and its metabolites in hepatocytes, it has been suggested that micromolar tissue concentrations are reached (Hurst and Waxman, 2004). Populations exposed to high doses of DEHP in these extreme cases, relevant to the doses used in our presented studies, may be subject to additional risk when receiving pharmaceutical doses of glucocorticoids which are given routinely in cancer therapies, to lessen the effects of mechanical ventilation or ECMO, or even in times of stress when circulating glucocorticoid levels spike (Griffin et al., 2004; Halliday, 2004; Heggen et al., 2004; Sapolsky et al., 2000; Thebaud et al., 2001; Wallace and Cidlowski, 2002).
Our findings implicate DEHP and MEHP as being involved in induction of enhanced steroid metabolism with concomitant glucocorticoid treatment. This presents a two-pronged effect on steroid hormone levels when you consider the previously established role of these phthalates in decreasing steroidogenesis pathways. The benefits to severely ill individuals of the previously described procedures, leading to high serum levels of DEHP, clearly exceed the health risks associated with DEHP exposure. Although no studies to date have investigated the effects of concomitant exposure to glucocorticoids and phthalates in humans or animal models, our findings suggest this is an area future studies should explore. Contributing to the effects we have observed by DEHP and MEHP may be the release of an endogenous PXR ligand or the production of MEHP oxidative metabolites that are PXR ligands. Additionally, the alteration of drug metabolism as a result of CYP enzyme induction may be responsible for changes in the efficacy and toxicity of pharmaceutical drugs as well as detrimental drug–drug interactions. The data presented here advance our understanding of how concomitant exposure to glucocorticoids and phthalate esters may alter the expression level and function of CYP3A4, a key player in phase I metabolism.
|
FUNDING |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
U.S. Army grant (project # DAMD 17-00-2-008) to T.M.C; and National Institute of Environmental Health Sciences Training grant (ES7046) to B.C.
|
ACKNOWLEDGMENTS |
|---|
We would like to thank Yan Cao, Edward Croom, and the late Dr Randy Rose for their technical assistance with this research. We would also like to thank Dr Consuello Arellano for her help with the statistical analysis and Dr Gerald LeBlanc, Dr Ernest Hodgson, Dr Seth Kullman, and Dr Elizabeth MacKenzie for their invaluable assistance in preparation of the manuscript.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Albro PW, Thomas RO. Enzymatic hydrolysis of di-(2-ethylhexyl) phthalate by lipases. Biochim. Biophys. Acta (1973) 306:380–390.[Medline]
Bagel-Boithias S, Sautou-Miranda V, Bourdeaux D, Tramier V, Boyer A, Chopineau J. Leaching of diethylhexyl phthalate from multilayer tubing into etoposide infusion solutions. Am. J. Health Syst. Pharm. (2005) 62:182–188.
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.
Blumberg B, Sabbagh W Jr, Juguilon H, Bolado J Jr, van Meter CM, Ong ES, Evans RM. SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev. (1998) 12:3195–3205.
Braissant O, Foufelle F, Scotto C, Dauca M, Wahli W. Differential expression of peroxisome proliferator-activated receptors (PPARs): Tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinology (1996) 137:354–366.[Abstract]
Brien TG. Free cortisol in human plasma. Horm. Metab. Res. (1980) 12:643–650.[Web of Science][Medline]
Fayer JL, Petullo DM, Ring BJ, Wrighton SA, Ruterbories KJ. A novel testosterone 6 beta-hydroxylase activity assay for the study of CYP3A-mediated metabolism, inhibition, and induction in vitro. J. Pharmacol. Toxicol. Methods (2001) 46:117–123.[CrossRef][Web of Science][Medline]
FDA. Safety assessment of di(2-ethylhexyl)phthalate (DEHP) released from PVC medical devices (2004) Rockville, MD: Center for Devices and Radiological Health, U.S. Food and Drug Administration.
Foster PM, Mylchreest E, Gaido KW, Sar M. Effects of phthalate esters on the developing reproductive tract of male rats. Hum. Reprod. Update (2001) 7:231–235.
Frederiksen H, Skakkebaek NE, Andersson AM. Metabolism of phthalates in humans. Mol. Nutr. Food Res. (2007) 51:899–911.[CrossRef][Web of Science][Medline]
Griffin MP, Wooldridge P, Alford BA, McIlhenny J, Ksenich RA. Dexamethasone therapy in neonates treated with extracorporeal membrane oxygenation. J. Pediatr. (2004) 144:296–300.[CrossRef][Web of Science][Medline]
Gross-Steinmeyer K, Stapleton PL, Tracy JH, Bammler TK, Lehman T, Strom SC, Eaton DL. Influence of Matrigel-overlay on constitutive and inducible expression of nine genes encoding drug-metabolizing enzymes in primary human hepatocytes. Xenobiotica (2005) 35:419–438.[CrossRef][Web of Science][Medline]
Halliday HL. Postnatal steroids and chronic lung disease in the newborn. Paediatr. Respir. Rev. (2004) 5(Suppl. A):S245–S248.[CrossRef][Medline]
Heggen JA, Fortenberry JD, Tanner AJ, Reid CA, Mizzell DW, Pettignano R. Systemic hypertension associated with venovenous extracorporeal membrane oxygenation for pediatric respiratory failure. J. Pediatr. Surg. (2004) 39:1626–1631.[CrossRef][Web of Science][Medline]
Hurst CH, Waxman DJ. Environmental phthalate monoesters activate pregnane X receptor-mediated transcription. Toxicol. Appl. Pharmacol. (2004) 199:266–274.[CrossRef][Web of Science][Medline]
Huss JM, Kasper CB. Nuclear receptor involvement in the regulation of rat cytochrome P450 3A23 expression. J. Biol. Chem. (1998) 273:16155–16162.
Huss JM, Kasper CB. Two-stage glucocorticoid induction of CYP3A23 through both the glucocorticoid and pregnane X receptors. Mol. Pharmacol. (2000) 58:48–57.
Huss JM, Wang SI, Astrom A, McQuiddy P, Kasper CB. Dexamethasone responsiveness of a major glucocorticoid-inducible CYP3A gene is mediated by elements unrelated to a glucocorticoid receptor binding motif. Proc. Natl. Acad. Sci. U. S. A. (1996) 93:4666–4670.
Kavlock R, Barr D, Boekelheide K, Breslin W, Breysse P, Chapin R, Gaido K, Hodgson E, Marcus M, Shea K, Williams P. NTP-CERHR expert panel update on the reproductive and developmental toxicity of di(2-ethylhexyl) phthalate. Reprod. Toxicol. (2006) 22:291–399.[CrossRef][Web of Science][Medline]
Kim HS, Saito K, Ishizuka M, Kazusaka A, Fujita S. Short period exposure to di-(2-ethylhexyl) phthalate regulates testosterone metabolism in testis of prepubertal rats. Arch. Toxicol. (2003) 77:446–451.[CrossRef][Web of Science][Medline]
Kim SC, Yoon HJ, Lee JW, Yu J, Park ES, Chi SC. Investigation of the release behavior of DEHP from infusion sets by paclitaxel-loaded polymeric micelles. Int. J. Pharm. (2005) 293:303–310.[CrossRef][Web of Science][Medline]
Kliewer SA, Goodwin B, Willson TM. The nuclear pregnane X receptor: A key regulator of xenobiotic metabolism. Endocr. Rev. (2002) 23:687–702.
Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, et al. An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell (1998) 92:73–82.[CrossRef][Web of Science][Medline]
Koch HM, Bolt HM, Angerer J. Di(2-ethylhexyl)phthalate (DEHP) metabolites in human urine and serum after a single oral dose of deuterium-labelled DEHP. Arch. Toxicol. (2004) 78:123–130.[CrossRef][Web of Science][Medline]
Koch HM, Bolt HM, Preuss R, Angerer J. New metabolites of di(2-ethylhexyl)phthalate (DEHP) in human urine and serum after single oral doses of deuterium-labelled DEHP. Arch. Toxicol. (2005) 79:367–376.[CrossRef][Web of Science][Medline]
Koch HM, Preuss R, Angerer J. Di(2-ethylhexyl)phthalate (DEHP): Human metabolism and internal exposure—An update and latest results. Int. J. Androl. (2006) 29:155–165. discussion 181–185.[CrossRef][Web of Science][Medline]
Kostrubsky VE, Ramachandran V, Venkataramanan R, Dorko K, Esplen JE, Zhang S, Sinclair JF, Wrighton SA, Strom SC. The use of human hepatocyte cultures to study the induction of cytochrome P-450. Drug Metab. Dispos. (1999) 27:887–894.
Latini G, De Felice C, Verrotti A. Plasticizers, infant nutrition and reproductive health. Reprod. Toxicol. (2004) 19:27–33.[CrossRef][Web of Science][Medline]
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.[CrossRef][Web of Science][Medline]
Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA. 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.[Web of Science][Medline]
Lehmann KP, Phillips S, Sar M, Foster PM, Gaido KW. Dose-dependent alterations in gene expression and testosterone synthesis in the fetal testes of male rats exposed to di (n-butyl) phthalate. Toxicol. Sci. (2004) 81:60–68.
Li AP, Kaminski DL, Rasmussen A. Substrates of human hepatic cytochrome P450 3A4. Toxicology (1995) 104:1–8.[CrossRef][Web of Science][Medline]
Loff S, Kabs F, Witt K, Sartoris J, Mandl B, Niessen KH, Waag KL. Polyvinylchloride infusion lines expose infants to large amounts of toxic plasticizers. J. Pediatr. Surg. (2000) 35:1775–1781.[CrossRef][Web of Science][Medline]
Maloney EK, Waxman DJ. trans-Activation of PPARalpha and PPARgamma by structurally diverse environmental chemicals. Toxicol. Appl. Pharmacol. (1999) 161:209–218.[CrossRef][Web of Science][Medline]
Masuyama H, Hiramatsu Y, Kunitomi M, Kudo T, MacDonald PN. Endocrine disrupting chemicals, phthalic acid and nonylphenol, activate pregnane X receptor-mediated transcription. Mol. Endocrinol. (2000) 14:421–428.
Pascussi JM, Drocourt L, Fabre JM, Maurel P, Vilarem MJ. Dexamethasone induces pregnane X receptor and retinoid X receptor-alpha expression in human hepatocytes: Synergistic increase of CYP3A4 induction by pregnane X receptor activators. Mol. Pharmacol. (2000) 58:361–372.
Pascussi JM, Drocourt L, Gerbal-Chaloin S, Fabre JM, Maurel P, Vilarem MJ. Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor. Eur. J. Biochem. (2001) 268:6346–6358.[Web of Science][Medline]
Plonait SL, Nau H, Maier RF, Wittfoht W, Obladen M. Exposure of newborn infants to di-(2-ethylhexyl)-phthalate and 2-ethylhexanoic acid following exchange transfusion with polyvinylchloride catheters. Transfusion (1993) 33:598–605.[CrossRef][Web of Science][Medline]
Quattrochi LC, Mills AS, Barwick JL, Yockey CB, Guzelian PS. A novel cis-acting element in a liver cytochrome P450 3A gene confers synergistic induction by glucocorticoids plus antiglucocorticoids. J. Biol. Chem. (1995) 270:28917–28923.
Raff H. Neonatal dexamethasone therapy: Short- and long-term consequences. Trends Endocrinol. Metab. (2004) 15:351–352.[CrossRef][Web of Science][Medline]
Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids—New mechanisms for old drugs. N. Engl. J. Med. (2005) 353:1711–1723.
Rhen T, Willingham E, Sakata JT, Crews D. Incubation temperature influences sex-steroid levels in juvenile red-eared slider turtles, Trachemys scripta, a species with temperature-dependent sex determination. Biol. Reprod. (1999) 61:1275–1280.
Sapolsky RM, Romero LM, Munck AU. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev. (2000) 21:55–89.
Schmid P, Schlatter C. Excretion and metabolism of di(2-ethylhexyl)phthalate in man. Xenobiotica (1985) 15:251–256.[Web of Science][Medline]
Schuetz EG, Guzelian PS. Induction of cytochrome P-450 by glucocorticoids in rat liver. II. Evidence that glucocorticoids regulate induction of cytochrome P-450 by a nonclassical receptor mechanism. J. Biol. Chem. (1984) 259:2007–2012.
Shea KM. Pediatric exposure and potential toxicity of phthalate plasticizers. Pediatrics (2003) 111:1467–1474.
Shiota K, Nishimura H. Teratogenicity of di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) in mice. Environ. Health Perspect. (1982) 45:65–70.[Web of Science][Medline]
Takeshita A, Inagaki K, Igarashi-Migitaka J, Ozawa Y, Koibuchi N. The endocrine disrupting chemical, diethylhexyl phthalate, activates MDR1 gene expression in human colon cancer LS174T cells. J. Endocrinol. (2006) 190:897–902.
Thebaud B, Lacaze-Masmonteil T, Watterberg K. Postnatal glucocorticoids in very preterm infants: "the good, the bad, and the ugly"? Pediatrics (2001) 107:413–415.
Tickner JA, Schettler T, Guidotti T, McCally M, Rossi M. Health risks posed by use of di-2-ethylhexyl phthalate (DEHP) in PVC medical devices: A critical review. Am. J. Ind. Med. (2001) 39:100–111.[CrossRef][Web of Science][Medline]
Usmani KA, Rose RL, Hodgson E. Inhibition and activation of the human liver microsomal and human cytochrome P450 3A4 metabolism of testosterone by deployment-related chemicals. Drug Metab. Dispos. (2003) 31:384–391.
Wallace AD, Cidlowski JA. Drug treatment: Corticosteroids. In: Encyclopedia of Cancer—Bertino JR, ed. (2002) 2nd ed. San Diego, CA: Academic Press. 23–31.
Ward JM, Peters JM, Perella CM, Gonzalez FJ. Receptor and nonreceptor-mediated organ-specific toxicity of di(2-ethylhexyl)phthalate (DEHP) in peroxisome proliferator-activated receptor alpha-null mice. Toxicol. Pathol. (1998) 26:240–246.
Waxman DJ, Attisano C, Guengerich FP, Lapenson DP. Human liver microsomal steroid metabolism: Identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme. Arch. Biochem. Biophys. (1988) 263:424–436.[CrossRef][Web of Science][Medline]
Zhang H, LeCulyse E, Liu L, Hu M, Matoney L, Zhu W, Yan B. Rat pregnane X receptor: Molecular cloning, tissue distribution, and xenobiotic regulation. Arch. Biochem. Biophys. (1999) 368:14–22.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Ott, G. Fricker, and B. Bauer Pregnane X Receptor (PXR) Regulates P-Glycoprotein at the Blood-Brain Barrier: Functional Similarities between Pig and Human PXR J. Pharmacol. Exp. Ther., April 1, 2009; 329(1): 141 - 149. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||