Di-n-Butyl Phthalate Activates Constitutive Androstane Receptor and Pregnane X Receptor and Enhances the Expression of Steroid-Metabolizing
http://www.100md.com
《毒物学科学杂志》
CIIT Centers for Health Research, Research Triangle Park, North Carolina
Department of Biomedical Sciences, University of Rhode Island, Kingston, Rhode Island
ABSTRACT
The plasticizer di-n-butyl phthalate (DBP) is a reproductive toxicant in rodents. Exposure to DBP in utero at high doses alters early reproductive development in male rats. Di-n-butyl phthalate also affects hepatic and extrahepatic enzymes. The objectives of this study were to determine the responsiveness of steroid-metabolizing enzymes in fetal liver to DBP and to investigate the potential of DBP to activate nuclear receptors that regulate the expression of liver enzymes. Pregnant Sprague-Dawley rats were orally dosed with DBP at levels of 10, 50, or 500 mg/kg/day from gestation days 12 to 19; maternal and fetal liver samples were collected on day 19 for analyses. Increased protein and mRNA levels of CYP 2B1, CYP 3A1, and CYP 4A1 were found in both maternal and fetal liver in the 500-mg dose group. Di-n-butyl phthalate at high doses also caused an increase in the mRNA of hepatic estrogen sulfotransferase and UDP-glucuronosyltransferase 2B1 in the dams but not in the fetuses. Xenobiotic induction of CYP3A1 and 2B1 is known to be mediated by the nuclear hormone receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). In vitro transcriptional activation assays showed that DBP activates both PXR and CAR. The main DBP metabolite, mono-butyl-phthalate (MBP) did not interact strongly with either CAR or PXR. These data indicate that hepatic steroid- and xenobiotic-metabolizing enzymes are susceptible to DBP induction at the fetal stage; such effects on enzyme expression are likely mediated by xenobiotic-responsive transcriptional factors, including CAR and PXR. Our study shows that DBP is broadly reactive with multiple pathways involved in maintaining steroid and lipid homeostasis.
Key Words: di-n-butyl phthalate; CYP2B; CYP3A; CAR; PXR; rat fetuses.
INTRODUCTION
Di-n-butyl phthalate (DBP) is a high-production-volume chemical used as a plasticizer and solvent in numerous consumer products. Humans are exposed to DBP through contaminated food and occupational sources (Kavlock et al., 2002). Data from the National Health and Nutrition Examination Survey (1999–2000) indicated that DBP exposure occurs in the general U.S. population, including children and women of child-bearing age (CDC, 2003); however, the estimated exposure levels are well under the Environmental Protection Agency reference dose (RfD) of 0.1 mg/kg/day (USEPA, 2005).
Di-n-butyl phthalate has been demonstrated to be a reproductive toxicant in laboratory animals (Kavlock et al., 2002). Male rats exposed to DBP at the perinatal stages develop adverse responses, including reduced anogenital distance, hypospadias, malformations of the epididymis and vas deferens, retention of thoracic nipples or areolae, and Leydig cell hyperplasia or abnormal formation of the seminiferous cord (Foster et al., 2001; Wine et al., 1997). These effects are proposed to manifest through an antiandrogenic mechanism, since testosterone production was reduced in the fetal testes after DBP exposure (Mylchreest et al., 1998, 2002; Shultz et al., 2001). In addition, the ability of many phthalates to interact with peroxisome proliferator–activated receptors (PPAR, , ) may also represent a mechanism for the phthalate-caused reproductive toxicity; but the evidence in this regard is not yet conclusive (Corton and Lapinskas, 2005).
In addition to the toxic effects in the reproductive tract, DBP exposure also causes an increase in liver weight and creates hepatic lesions (Marsman, 1995; Wine et al., 1997). The increase in liver organ weight is accompanied by enhanced total cytochrome P450 (CYP) enzyme activity (Walseth and Nilsen, 1986). Among the mediators for DBP-caused enzyme induction are PPARs, which are known to be transcriptional factors targeting P450 genes (Waxman, 1999; You, 2004). Di-n-butyl phthalate activates PPAR (Lapinskas et al., 2005) and causes changes in the expression of a number of PPAR-regulated genes (Fan et al., 1998; O'Brien et al., 2001; Wong and Gill, 2002). The main metabolite of DBP, mono-n-butyl phthalate (MBP), was shown to be inactive at both PPAR and PPAR (Hurst and Waxman, 2003; Lapinskas et al., 2005). The inability of MPB to activate the PPARs suggests a possibility that other transcriptional factors may be involved in the DBP-associated changes in hepatic enzyme expression.
Like PPAR, the constitutive active receptor (CAR) and the pregnane X receptor (PXR) are nuclear receptors that are highly enriched in the liver and that function as transcriptional regulators for a number of metabolic enzymes (reviewed in Handschin and Meyer, 2003; Wang and Negishi, 2003). Target genes for CAR and PXR include the families of CYP 2B, CYP 3A, and UDP glucuronosyltransferases (UGT) (Honkakoski et al., 1998; Lin and Wong, 2002; Wyde et al., 2003). These genes are involved in the metabolism of drugs, toxicants, and endogenous substances such as lipids, bile acids, and steroids (Mohan and Heyman, 2003). Changes of steroid metabolism and homeostasis may be an important component in the endocrine and reproductive toxicities of phthalates. The objectives of this study were to determine the responsiveness of steroid-metabolizing enzymes to DBP exposure and to establish relevant mechanisms. We evaluated the expression levels in fetal liver for CYP2B and 3A, UGT, and estrogen sulfotransferase (EST) in response to DPB exposure; we also investigated the potential of DBP to interact with nuclear receptors that regulate the expression of these enzymes. We found that hepatic CYP2B and CYP3A were inducible by DBP at the fetal stage, likely as the result of a mechanism of xenobiotic activation of nuclear receptors CAR and PXR. Such enzyme modulations suggest a potential for DBP to interfere with steroid and lipid homeostasis.
METHODS
Animals and treatment.
Time-mated Sprague-Dawley female rats were purchased from Charles River Laboratories (Raleigh, NC) and delivered on gestation day (GD) 0, the day that sperm was detected in the vaginal smear. Upon randomization into different treatment groups, the pregnant dams were housed individually in plastic cages with dry cellulose bedding (Shepherd Specialty Papers, Kalamazoo, MI). Rodent diet NIH-07 (Zeigler Brothers, Gardners, PA) and reverse-osmosis water were provided ad libitum. Animals were identified by ear tags and cage cards. The animal room was maintained within a temperature range of 22–25°C, relative humidity of 50 ± 10%, and 12-h light cycles (7:00–19:00). Body weight and food consumption of each dam were recorded on a twice-weekly schedule.
Dams were treated with DBP (Aldrich, Milwaukee, WI) by daily gavage in corn oil vehicle from GD 12 to GD 19. Di-n-butyl phthalate was administered at dose levels of 0, 10, 50, or 500 mg/kg/day. All dams were euthanized by CO2 asphyxiation on GD 19 at 2 h following the last dose. Fetuses were removed by cesarean section. All fetuses were euthanized by decapitation, and their sex was determined by internal examination of the reproductive organs. The fetal livers from male and female fetuses and liver tissue from the dams were snap-frozen in liquid nitrogen and stored separately at –80°C. For analyses performed for this report, liver samples were obtained from one male and one female fetus in each pregnant dam; four dams were included in each treatment group. Experimental details of this study were also described elsewhere (Lehmann et al., 2004).
Protein immunoblotting.
Immunoblotting was performed as previously described (You et al., 1999) for the cytochrome P450 enzymes CYP 3A1, 2B1, 1A1, and 4A and nuclear receptors CAR, PXR, aryl hydrocarbon receptor (AhR), and PPAR. For each treatment group, 4 samples, from fetuses of different maternal sources, were included for analysis in two separate blots. Total protein extracts from liver tissue were denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12% polyacrylamide. Proteins were transferred to nitrocellulose membranes; transfer efficiency and equal loading of different samples were confirmed by visual inspection of Ponceau Red staining. The membranes were then blocked for nonspecific binding, and incubated with polyclonal primary antibodies for CYP3A1, CYP2B1, CYP1A1, CYP4A, CAR, PXR, AhR, and PPAR. After incubation with primary antibody, membranes were incubated with horseradish peroxidase–linked anti-rabbit (CYP3A1, PXR, PPAR, and CAR) or anti-goat (CYP1A1, CYP2B1, and CYP 4A1) IgG secondary antibodies and visualized on film exposed to enhanced chemiluminescence (Hyperfilm-ECL, Amersham). Goat anti-rat polyclonal antibodies against rat CYP2B1 and CYP4A1 were obtained from Daiichi Pure Chemical Company (Tokyo, Japan). CYP3A1 antibodies were obtained from Research Diagnostics, Inc. (Flanders, NJ). CYP1A1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-PXR and anti-CAR antibodies were used as previously described (Wyde et al., 2003). Rabbit anti-PPAR antibody (H-98) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-AhR antibody was obtained from Affinity Bioreagents (Golden, CO).
The relative protein amounts in identified immunoblot bands were estimated by measuring the optical densities of the bands on exposed Autorad films, with the NIH ImageJ software (Rasband, 2005). The measurements were background adjusted and the values were statistically analyzed.
Quantitative RT-PCR.
To quantitate the amount of CYP 2B1 and 3A1 mRNA, cDNA was synthesized from total RNA isolated from liver tissue. Random hexamers and the Taqman reverse transcription reagents (PE Applied Biosystems, Foster City, CA) were used according to the manufacturer's protocol. The PCR primers were designed with Primer Express software (PE Applied Biosystems). The design parameters were as follows: low Tm = 60°C, high Tm = 64°C, optimum Tm = 62°, amplicon length = 80–150 bp, and primer length 20–24 bp, with an optimum of 22 bp.
The production of a single PCR product was confirmed by gel electrophoresis for each pair of PCR primers before quantification. Primer efficiency was determined according to the manufacturer's suggested protocol. Real-time quantitative PCR (Taqman) was performed on a 7700 PRISM Sequence Detector (Applied Biosystems), using either SYBR Green (for CYP2B1, CYP3A1, and PXR) or a probe sequence (for CAR, EST, and UGT2B1) according to the manufacturer's instructions, for quantification of relative gene expression (User Bulletin no. 2: P/N 4303859). GAPDH was used as a housekeeping gene for normalization. The primary and probe sequences are listed in Table 1.
PXR and CAR transactivation assays.
Transient transcriptional activation assays for CAR and PXR have been described elsewhere (Wyde et al., 2003). The assays for CAR and PXR transactivation assays were developed in different laboratories using two different cell lines. Both assays have been applied extensively and broadly (Yoshinari et al., 2001; Zhang et al., 1999); both cell lines contain the receptor heterodimer partner RXR and are known to support transcriptional activities of nuclear receptors. The choice of cell lines in this study therefore was not expected to affect the basic results. Briefly, COS-7 cells were used for transient transfection assay of PXR activation. Pregnane X receptor transfection was conducted by lipofection with LipofectAMINE (Gibco/BRL) and 100 ng of rat PXR plasmid, 100 ng of reporter plasmid (pGL-3 SV40 firefly luciferase containing two copies of rat PXR response element [IR6 and DR6] in the promoter region), and 10 ng of pRL-TK plasmid containing Renilla Luciferase. Assays to determine the activation of CAR were based on co-transfection of HepG2 cells with the rat CAR expression vector (Yoshinari et al., 2001), luciferase reporter plasmid [(NR1)5-tk-Luc], and pRL-SV40 Renilla Luciferase (Wyde et al., 2003). Co-transfection of CAR and reporter vectors used TransIT-LT1 reagents (Mirus, Madison, WI). For both the PXR and the CAR assays, transfected cells were cultured for 24 h before being treated with DBP and MBP at various concentrations for an additional 24 h, and the reporter enzyme activities were assayed with a Dual-Luciferase Reporter Assay System (Promega, Madison, WI), in which the Renilla Luciferase was used as transfection control. The firefly luciferase reporter activity was normalized based on the transfection control. The treatment data were then derived by normalizing to DMSO control values.
Statistics.
All data are presented as means ± standard deviation. Significant differences were determined by analysis of variance (ANOVA) and Dunnett's test (p < 0.05).
RESULTS
A basal level expression of hepatic CYP 3A1 and 2B1 proteins was detected through immunoblotting in untreated male and female Sprague-Dawley rat fetuses at GD 19 (Fig. 1A–1D). Treatment with DBP from GD 12 to 19 at the maternal daily gavage dose of 500 mg/kg (but not at 10 mg/kg and 50 mg/kg) increased significantly (p < 0.05) the hepatic CYP 2B1 protein in both male and female fetuses (Fig. 1A and 1B). The level of hepatic 3A1 protein was not affected by in utero exposure to DBP in either male or female fetuses (Fig. 1C and 1D). A basal expression of CYP4A1 was also detected in fetal liver; this CYP isoform is significantly induced by DBP treatment in both female and male fetuses at the 500 mg/kg dose level (Fig. 1E and 1F). In several cases (female and male CYP2B1 and male CYP4A1), the amounts of enzyme proteins seemed to decrease from the control group to the 5 mg/kg and 50 mg/kg DBP groups before they became significantly induced at the 500 mg/kg level. These downward trends were not statistically significant.
Nuclear receptor CAR and PXR proteins were detectable in the livers of both male and female rat fetuses at GD 19 (Fig. 2). No difference in CAR protein level was observed in males or females between control and DBP-treated fetuses (Fig. 2A and 2B). The level of PXR protein in GD 19 fetal liver was increased in both male and female rats in the 500 mg/kg group (Fig. 2C and 2D), whereas the doses of 10 and 50 mg DBP/kg had no effect in this regard. The protein levels of hepatic PPAR and CYP4A were also increased in both male and female fetuses in the 500 mg/kg DBP group (data not shown).
The proteins of CAR, PXR, CYP 2B1, and CYP 3A1 were detected in liver samples of the pregnant dams (data not shown). The basal levels of these proteins in the maternal liver were greatly enhanced beyond those observed in the fetal liver. Di-n-butyl phthalate exposure did not cause an appreciable change in the levels of CAR and PXR proteins; however, the amounts of CYP2B1 and 3A1 were moderately increased in the 500 mg/kg dose group (data not shown).
Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) was performed to determine the amounts of mRNA of CAR, PXR, CYP 2B1, CYP 3A1, and the steroid-conjugating enzymes UGT2B1 and EST. Liver mRNA levels of both CYP 2B1 and 3A1 were increased twofold in the dams exposed to 500 mg DBP/kg compared to the controls (Fig. 3). In addition, mRNA levels of EST were increased twofold and threefold at the 50 and 500 mg/kg doses, respectively, whereas the CAR mRNA was increased fourfold at the 500 mg/kg dose level. In the male fetuses, hepatic CYP2B1 mRNA was markedly increased by the 500 mg/kg DBP treatment, which also increased the PXR mRNA (Fig. 3). Female fetuses showed a response pattern identical to that of male fetuses, with both CYP2B1 and PXR mRNA increases at similar dose–response magnitudes (data not shown). No treatment-related differences were detected in mRNA expression of CYP 3A1, UGT2B1, EST, or CAR in fetal liver.
The ability of DBP to activate PXR and CAR was investigated in cell lines transiently transfected with expression plasmids for the receptors and luciferase reporters. Constitutive androstane receptor activation was tested with rat CAR-transfected HepG2 cells and a dual luciferase reporter assay, whereas PXR activation was tested with PXR-transfected COS-7 cells and a dual luciferase reporter assay. Both systems have been used in numerous studies, and both were proven to be sensitive and specific assay tools for detecting the activation of the respective receptors (Wyde et al., 2003; Yoshinari et al., 2001; Zhang et al., 1999). In the CAR assay, we used androstenol (an inverse agonist) (Forman et al., 1998) to repress the constitutive CAR activity and 1,1-dichloro-2,2-bis (p-chlorophenyl)ethylene (DDE) as a positive control (Wyde et al., 2003). Di-n-butyl phthalate treatment did not result in CAR activation; rather, it caused slight inhibition of the CAR transcriptional activity (Fig. 4A). Cells treated with the inverse agonist androstenol (4 μM) demonstrated approximately an 80% inhibition in CAR transcriptional activity, and this inhibition was reversed by DBP treatment in a dose-dependent manner in the concentration range of 5 to 50 μM (Fig. 4B).
For PXR assays, we used pregnenolone 16--carbonitrile (PCN) and rifampicin as rat and human PXR positive controls, respectively (Zhang et al., 1999). The PXR transactivation assay demonstrated an ability of DBP to activate both rat and human PXR (Fig. 5). Activation of the rat PXR occurred at a lower dose (5 μM) than that for human PXR (20 μM). The peak activation of rat PXR occurred at 20 μM of DBP; that of human PXR, at 50 μM.
The ability of MBP, the main hydrolytic product of DBP, to activate PXR and CAR was also investigated. Mono-butyl-phthalate treatment did not increase the CAR receptor activity. nor did it reverse the repression of CAR by androstenol (Fig. 6A and 6B). Mono-butyl-phthalate treatment did, however, increase rat (but not human) PXR activation approximately twofold at the concentration of 50 μM (Fig 6C and 6D); this increase, however, was much smaller than the 8-fold induction caused by DBP (Fig. 5A).
DISCUSSION
At the fetal stage, the developing animal is particularly sensitive to reproductive effects of DBP, which can manifest at maternal doses that are without apparent effect on the dam (Mylchreest et al., 1998, 1999; Parks et al., 2000). In fetal testes, DBP exposure represses the genes involved in steroidogenesis, resulting in reduced androgen production (Lehmann et al., 2004; Mylchreest et al., 2002; Parks et al., 2000). The current study demonstrated that fetal liver, like fetal testis, is also susceptible to DBP through maternal exposure. We also demonstrated that DBP activates CAR and PXR, in addition to being a known activator of PPAR; these nuclear receptors are likely among the key regulators for DBP effects on liver enzymes.
The no-observable-adverse-effect level (NOAEL) of DBP-caused reproductive development effects in male rats was established at the 50 mg/kg maternal gavage dose (Mylchreest et al., 2000), whereas the lowest-observable-adverse-effect level (LOAEL) of DBP-caused inhibition on the expression of testicular steroidogenic genes was determined to be the 50 mg/kg maternal gavage dose (Lehmann et al., 2004). The current study detected DBP effects on the metabolic apparatus in the maternal and fetal liver at the 500 mg/kg dose level in proteins (CYP2B1 and PXR) and at the 50 mg/kg dose level in mRNA (CYP3A1, CYP2B1, and EST in the dams and CYP2B1 and PXR in the fetuses). The Lehmann et al. study (2004) demonstrated coordinated changes in gene expression of key testicular steroidogenic factors and testosterone production at the 50 mg/kg dose level. We do not know whether the similar sensitivities to DBP treatment of hepatic-metabolizing enzymes and testicular steroidogenic enzymes are based on shared mechanisms in the liver and testis.
Phthalates belong to a class of peroxisome proliferator chemicals. Exposure to those chemicals evokes a set of pleiotropic responses that include hepatocellular hypertrophy, hyperplasia, and induction of metabolic enzymes in rodent liver (Lock et al., 1989); these effects are known to be mediated mainly through PPAR (Reddy and Hashimoto, 2001). The ability of DBP to activate PPAR was demonstrated in an in vitro reporter gene transactivation assay (Lapinskas et al., 2005). This activation of PPAR explains a DBP-caused increase in hepatic CYP4A expression (Lapinskas et al., 2005), since CYP4A is a well-characterized target gene of PPAR (Lee et al., 1995; Ripp et al., 2002). As expected, the present study found that DBP exposure induced hepatic CYP4A1, and this induction, presumably mediated by PPAR, was operative at the fetal stage.
However, PPAR activation cannot explain the induction of CYP2B1 and 3A1 by DBP in the present study. In PPAR-null mouse, CYP3A11, a mouse homolog of the rat CYP3A1 gene, is inducible by xenobiotics (Ripp et al., 2002). In contrast, CYP2B was not inducible in CAR-null mouse, and CYP3A was not inducible in PXR-null mouse (Sonoda and Evans, 2003). Constitutive androstane receptor and PXR regulate hepatic genes, including the CYP2B and 3A families (Wang and Negishi, 2003; Wei et al., 2002). Thus, activation of PXR and CAR, but not PPAR, was likely required for the CYP3A1 and 2B1 induction in fetal rat liver by DBP. Indeed, DBP was shown to enhance the expression of hepatic CYP2B and 3A, whereas the PPAR agonist Wy-14,643 did not (Fan et al., 2004), further supporting that activating PXR and CAR, rather than PPAR, is responsible for the DPB effects. We have demonstrated in the present study that DBP interacts directly with CAR and PXR; such interactions are highly likely to be the mechanisms for DBP induction of genes in the CYP2B and 3A families (Wang and Negishi, 2003; Wei et al., 2002). The manner in which DBP activates PXR resembles the activation of PXR by DDE (Wyde et al., 2003). Di-n-butyl phthalate interacts differently with CAR than with PXR. Although DBP did not change the constitutive activity of CAR, it reversed the androstenol-imposed CAR repression; this type of CAR activation has been shown for other CAR activators as well (Blizard et al., 2001). Although we did not examine enzyme activities in these experiments, transcriptional increase of the hepatic CYP enzymes is known to correlate well with their protein levels and catalytic activities (Fan et al., 2004; Wyde et al., 2003; You et al., 1999).
We noted that, at the lower doses of DPB used in this study (5 mg/kg and 50 mg/kg), the protein amounts of several CYP isoforms seemed to be reduced, contrary to inductions at 500 mg/kg dose level. Similar reduction of CYP3A1 was previously reported to be associated with treatment with DDE and mifepristone (RU486), both PXR activators (Schuetz et al., 2000; Wyde et al., 2003). One potential mechanism for DBP to have such a seemingly biphasic effect on CYP expression is the mediation of glucocorticoid actions. The glucocorticoid receptor (GR) is essential for both basal and stimulated expression of CYP2B (Schuetz et al., 2000). Glucocorticoid-receptor–enhanced CYP expression is not mediated through cis-acting element but through complex protein–protein interactions (Honkakoski and Negishi, 2000). Activation of GR results in enhanced expression of PXR and RXR (Pascussi et al., 2000); the latter is the heterodimer partner of both PXR and CAR. Di-n-butyl phthalate may thus act either through an inhibition on steroidogenesis (reducing glucocorticoid level) or through displacement of endogenous activators at PXR (and possibly other CYP-regulating nuclear receptors) to cause reduction in certain CYP isoforms at specific doses.
Phthalic acid was reported to promote PXR interaction with steroid hormone receptor coactivator-1 (SRC-1), to increase PXR transcriptional activity in reporter gene assay, and to induce CYP3A1 in adult male rat liver (Masuyama et al., 2000). In the current study, we demonstrated that fetal liver is also susceptible to the effects of phthalates in regard to metabolic enzyme induction. We detected the proteins of CAR, PXR, AhR, and PPAR in fetal liver tissue. These receptors function as ligand-responsive transcriptional factors regulating hepatic induction of CYP2B1, 3A1, 1A1, and 4A1 in the rat. Constitutive androstane receptor and PXR also cross-regulate the CYP2B1 and 3A1 genes (Honkakoski et al., 2003). Detection of these receptor regulators of hepatic CYP enzymes at the fetal stage suggests physiological roles of these receptors in sensing and regulating the fetal environment. Although the CYP enzymes are regulated by ligand activation of these receptors, treatment-caused changes in receptor expression level may also play a role in controlling target gene expression. By examining the relationship between nuclear receptor expression and their target enzyme expression, we found no consistent coupling between increase in receptor expression and increase in enzyme expression.
A number of hepatocyte-enriched transcription factors are essential in coordinating gene expression during fetal liver differentiation (Cereghini, 1996); the differentiation process is necessary for the developing liver to acquire metabolic capacity. Hepatocyte nuclear factor-4 (HNF4) is a transcriptional coregulator of CAR and PXR for the human CYP3A4 gene (Tirona et al., 2003). In addition, HNF4 is a transcriptional factor for the PXR gene in fetal hepatocytes (Kamiya et al., 2003). In the present study, DBP treatment enhanced the expression of PXR and PPAR. Whether DBP interacts with hepatic transcriptional factors such as HNF4 remains to be seen; such interaction, if exists, would provide a mechanism for DBP-caused changes in the expression of PXR and PPAR.
Di-n-butyl phthalate toxicity is attributed in large part to MBP, a major hydrolysis product of DBP in vivo (Kavlock et al., 2002). In contrast to the ability of DBP to activate CAR and PXR, MBP showed little or no ability to activate these receptors. Similarly, MBP activation of PPAR was reported to be insignificant (Lapinskas et al., 2005). The fact that only very high doses of DBP caused induction of CYP enzymes suggests the possibility that a portion of the parent compound may escape the initial metabolism at high doses, that it may reach fetal liver cells, and that it may activate the corresponding receptors. Another possibility is that MBP may alter gene expression of CYP enzymes through mechanisms that are receptor independent.
In addition to CYP enzymes, CAR and PXR also regulate the expression of conjugating enzymes in the families of glutathione S-transferases (GST), UDP-glucuronosyltransferases (UGT), sulfotransferases (SULT), and multidrug-resistance–associated proteins (MRP) (Maglich et al., 2002; Xie et al., 2003). Although we detected only a slight change of UGT2B1 mRNA in dam liver (but not in fetuses), we found greater changes (over twofold and threefold at 50 and 500 mg/kg doses) for the expression of estrogen sulfotransferase. The induction of sulfotransferase is likely a consequence of activating CAR, instead of PXR, because the rat CAR activator TCPOBOP, but not the rat PXR activator PCN, was shown to be associated with regulating sulfotransferase expression (Maglich et al., 2002). The effects of phthalate on UGTs and SULTs are important, because the conjugating reactions catalyzed by these enzymes render the many endogenous molecules and chemical metabolites highly hydrophilic and easily excreted from the body (You, 2004). The lack of response in conjugating enzyme expression to DBP in the fetal liver suggests that a change in these conjugation pathways is not a significant factor in regulating hormone activities in the fetuses.
Although there may be implications with regard to developmental dysregulation, the significance of fetal liver effects caused by DBP after maternal exposure remains to be adequately appraised. The diverse nature of DBP interaction with nuclear receptors PXR, CAR, and PPAR, among possible others, and the extensive involvement of these nuclear receptors in regulating numerous metabolic pathways suggest broad potentials of DBP modulation on the metabolism of lipids, steroids, and other biological processes, including lipid homeostasis, cholesterol metabolism, and steroidogenesis in the gonads.
ACKNOWLEDGMENTS
This research was funded by the American Chemistry Council Long-Range Research Initiative (to L.Y.) and a National Institutes of Health grant (R01-GM61988) (to B.Y.). We thank Drs. Susan Borghoff and Kamin Johnson for reading the manuscript and Dr. Barbara Kuyper for editorial review. Conflict of interest: none declared.
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Department of Biomedical Sciences, University of Rhode Island, Kingston, Rhode Island
ABSTRACT
The plasticizer di-n-butyl phthalate (DBP) is a reproductive toxicant in rodents. Exposure to DBP in utero at high doses alters early reproductive development in male rats. Di-n-butyl phthalate also affects hepatic and extrahepatic enzymes. The objectives of this study were to determine the responsiveness of steroid-metabolizing enzymes in fetal liver to DBP and to investigate the potential of DBP to activate nuclear receptors that regulate the expression of liver enzymes. Pregnant Sprague-Dawley rats were orally dosed with DBP at levels of 10, 50, or 500 mg/kg/day from gestation days 12 to 19; maternal and fetal liver samples were collected on day 19 for analyses. Increased protein and mRNA levels of CYP 2B1, CYP 3A1, and CYP 4A1 were found in both maternal and fetal liver in the 500-mg dose group. Di-n-butyl phthalate at high doses also caused an increase in the mRNA of hepatic estrogen sulfotransferase and UDP-glucuronosyltransferase 2B1 in the dams but not in the fetuses. Xenobiotic induction of CYP3A1 and 2B1 is known to be mediated by the nuclear hormone receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). In vitro transcriptional activation assays showed that DBP activates both PXR and CAR. The main DBP metabolite, mono-butyl-phthalate (MBP) did not interact strongly with either CAR or PXR. These data indicate that hepatic steroid- and xenobiotic-metabolizing enzymes are susceptible to DBP induction at the fetal stage; such effects on enzyme expression are likely mediated by xenobiotic-responsive transcriptional factors, including CAR and PXR. Our study shows that DBP is broadly reactive with multiple pathways involved in maintaining steroid and lipid homeostasis.
Key Words: di-n-butyl phthalate; CYP2B; CYP3A; CAR; PXR; rat fetuses.
INTRODUCTION
Di-n-butyl phthalate (DBP) is a high-production-volume chemical used as a plasticizer and solvent in numerous consumer products. Humans are exposed to DBP through contaminated food and occupational sources (Kavlock et al., 2002). Data from the National Health and Nutrition Examination Survey (1999–2000) indicated that DBP exposure occurs in the general U.S. population, including children and women of child-bearing age (CDC, 2003); however, the estimated exposure levels are well under the Environmental Protection Agency reference dose (RfD) of 0.1 mg/kg/day (USEPA, 2005).
Di-n-butyl phthalate has been demonstrated to be a reproductive toxicant in laboratory animals (Kavlock et al., 2002). Male rats exposed to DBP at the perinatal stages develop adverse responses, including reduced anogenital distance, hypospadias, malformations of the epididymis and vas deferens, retention of thoracic nipples or areolae, and Leydig cell hyperplasia or abnormal formation of the seminiferous cord (Foster et al., 2001; Wine et al., 1997). These effects are proposed to manifest through an antiandrogenic mechanism, since testosterone production was reduced in the fetal testes after DBP exposure (Mylchreest et al., 1998, 2002; Shultz et al., 2001). In addition, the ability of many phthalates to interact with peroxisome proliferator–activated receptors (PPAR, , ) may also represent a mechanism for the phthalate-caused reproductive toxicity; but the evidence in this regard is not yet conclusive (Corton and Lapinskas, 2005).
In addition to the toxic effects in the reproductive tract, DBP exposure also causes an increase in liver weight and creates hepatic lesions (Marsman, 1995; Wine et al., 1997). The increase in liver organ weight is accompanied by enhanced total cytochrome P450 (CYP) enzyme activity (Walseth and Nilsen, 1986). Among the mediators for DBP-caused enzyme induction are PPARs, which are known to be transcriptional factors targeting P450 genes (Waxman, 1999; You, 2004). Di-n-butyl phthalate activates PPAR (Lapinskas et al., 2005) and causes changes in the expression of a number of PPAR-regulated genes (Fan et al., 1998; O'Brien et al., 2001; Wong and Gill, 2002). The main metabolite of DBP, mono-n-butyl phthalate (MBP), was shown to be inactive at both PPAR and PPAR (Hurst and Waxman, 2003; Lapinskas et al., 2005). The inability of MPB to activate the PPARs suggests a possibility that other transcriptional factors may be involved in the DBP-associated changes in hepatic enzyme expression.
Like PPAR, the constitutive active receptor (CAR) and the pregnane X receptor (PXR) are nuclear receptors that are highly enriched in the liver and that function as transcriptional regulators for a number of metabolic enzymes (reviewed in Handschin and Meyer, 2003; Wang and Negishi, 2003). Target genes for CAR and PXR include the families of CYP 2B, CYP 3A, and UDP glucuronosyltransferases (UGT) (Honkakoski et al., 1998; Lin and Wong, 2002; Wyde et al., 2003). These genes are involved in the metabolism of drugs, toxicants, and endogenous substances such as lipids, bile acids, and steroids (Mohan and Heyman, 2003). Changes of steroid metabolism and homeostasis may be an important component in the endocrine and reproductive toxicities of phthalates. The objectives of this study were to determine the responsiveness of steroid-metabolizing enzymes to DBP exposure and to establish relevant mechanisms. We evaluated the expression levels in fetal liver for CYP2B and 3A, UGT, and estrogen sulfotransferase (EST) in response to DPB exposure; we also investigated the potential of DBP to interact with nuclear receptors that regulate the expression of these enzymes. We found that hepatic CYP2B and CYP3A were inducible by DBP at the fetal stage, likely as the result of a mechanism of xenobiotic activation of nuclear receptors CAR and PXR. Such enzyme modulations suggest a potential for DBP to interfere with steroid and lipid homeostasis.
METHODS
Animals and treatment.
Time-mated Sprague-Dawley female rats were purchased from Charles River Laboratories (Raleigh, NC) and delivered on gestation day (GD) 0, the day that sperm was detected in the vaginal smear. Upon randomization into different treatment groups, the pregnant dams were housed individually in plastic cages with dry cellulose bedding (Shepherd Specialty Papers, Kalamazoo, MI). Rodent diet NIH-07 (Zeigler Brothers, Gardners, PA) and reverse-osmosis water were provided ad libitum. Animals were identified by ear tags and cage cards. The animal room was maintained within a temperature range of 22–25°C, relative humidity of 50 ± 10%, and 12-h light cycles (7:00–19:00). Body weight and food consumption of each dam were recorded on a twice-weekly schedule.
Dams were treated with DBP (Aldrich, Milwaukee, WI) by daily gavage in corn oil vehicle from GD 12 to GD 19. Di-n-butyl phthalate was administered at dose levels of 0, 10, 50, or 500 mg/kg/day. All dams were euthanized by CO2 asphyxiation on GD 19 at 2 h following the last dose. Fetuses were removed by cesarean section. All fetuses were euthanized by decapitation, and their sex was determined by internal examination of the reproductive organs. The fetal livers from male and female fetuses and liver tissue from the dams were snap-frozen in liquid nitrogen and stored separately at –80°C. For analyses performed for this report, liver samples were obtained from one male and one female fetus in each pregnant dam; four dams were included in each treatment group. Experimental details of this study were also described elsewhere (Lehmann et al., 2004).
Protein immunoblotting.
Immunoblotting was performed as previously described (You et al., 1999) for the cytochrome P450 enzymes CYP 3A1, 2B1, 1A1, and 4A and nuclear receptors CAR, PXR, aryl hydrocarbon receptor (AhR), and PPAR. For each treatment group, 4 samples, from fetuses of different maternal sources, were included for analysis in two separate blots. Total protein extracts from liver tissue were denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12% polyacrylamide. Proteins were transferred to nitrocellulose membranes; transfer efficiency and equal loading of different samples were confirmed by visual inspection of Ponceau Red staining. The membranes were then blocked for nonspecific binding, and incubated with polyclonal primary antibodies for CYP3A1, CYP2B1, CYP1A1, CYP4A, CAR, PXR, AhR, and PPAR. After incubation with primary antibody, membranes were incubated with horseradish peroxidase–linked anti-rabbit (CYP3A1, PXR, PPAR, and CAR) or anti-goat (CYP1A1, CYP2B1, and CYP 4A1) IgG secondary antibodies and visualized on film exposed to enhanced chemiluminescence (Hyperfilm-ECL, Amersham). Goat anti-rat polyclonal antibodies against rat CYP2B1 and CYP4A1 were obtained from Daiichi Pure Chemical Company (Tokyo, Japan). CYP3A1 antibodies were obtained from Research Diagnostics, Inc. (Flanders, NJ). CYP1A1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-PXR and anti-CAR antibodies were used as previously described (Wyde et al., 2003). Rabbit anti-PPAR antibody (H-98) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-AhR antibody was obtained from Affinity Bioreagents (Golden, CO).
The relative protein amounts in identified immunoblot bands were estimated by measuring the optical densities of the bands on exposed Autorad films, with the NIH ImageJ software (Rasband, 2005). The measurements were background adjusted and the values were statistically analyzed.
Quantitative RT-PCR.
To quantitate the amount of CYP 2B1 and 3A1 mRNA, cDNA was synthesized from total RNA isolated from liver tissue. Random hexamers and the Taqman reverse transcription reagents (PE Applied Biosystems, Foster City, CA) were used according to the manufacturer's protocol. The PCR primers were designed with Primer Express software (PE Applied Biosystems). The design parameters were as follows: low Tm = 60°C, high Tm = 64°C, optimum Tm = 62°, amplicon length = 80–150 bp, and primer length 20–24 bp, with an optimum of 22 bp.
The production of a single PCR product was confirmed by gel electrophoresis for each pair of PCR primers before quantification. Primer efficiency was determined according to the manufacturer's suggested protocol. Real-time quantitative PCR (Taqman) was performed on a 7700 PRISM Sequence Detector (Applied Biosystems), using either SYBR Green (for CYP2B1, CYP3A1, and PXR) or a probe sequence (for CAR, EST, and UGT2B1) according to the manufacturer's instructions, for quantification of relative gene expression (User Bulletin no. 2: P/N 4303859). GAPDH was used as a housekeeping gene for normalization. The primary and probe sequences are listed in Table 1.
PXR and CAR transactivation assays.
Transient transcriptional activation assays for CAR and PXR have been described elsewhere (Wyde et al., 2003). The assays for CAR and PXR transactivation assays were developed in different laboratories using two different cell lines. Both assays have been applied extensively and broadly (Yoshinari et al., 2001; Zhang et al., 1999); both cell lines contain the receptor heterodimer partner RXR and are known to support transcriptional activities of nuclear receptors. The choice of cell lines in this study therefore was not expected to affect the basic results. Briefly, COS-7 cells were used for transient transfection assay of PXR activation. Pregnane X receptor transfection was conducted by lipofection with LipofectAMINE (Gibco/BRL) and 100 ng of rat PXR plasmid, 100 ng of reporter plasmid (pGL-3 SV40 firefly luciferase containing two copies of rat PXR response element [IR6 and DR6] in the promoter region), and 10 ng of pRL-TK plasmid containing Renilla Luciferase. Assays to determine the activation of CAR were based on co-transfection of HepG2 cells with the rat CAR expression vector (Yoshinari et al., 2001), luciferase reporter plasmid [(NR1)5-tk-Luc], and pRL-SV40 Renilla Luciferase (Wyde et al., 2003). Co-transfection of CAR and reporter vectors used TransIT-LT1 reagents (Mirus, Madison, WI). For both the PXR and the CAR assays, transfected cells were cultured for 24 h before being treated with DBP and MBP at various concentrations for an additional 24 h, and the reporter enzyme activities were assayed with a Dual-Luciferase Reporter Assay System (Promega, Madison, WI), in which the Renilla Luciferase was used as transfection control. The firefly luciferase reporter activity was normalized based on the transfection control. The treatment data were then derived by normalizing to DMSO control values.
Statistics.
All data are presented as means ± standard deviation. Significant differences were determined by analysis of variance (ANOVA) and Dunnett's test (p < 0.05).
RESULTS
A basal level expression of hepatic CYP 3A1 and 2B1 proteins was detected through immunoblotting in untreated male and female Sprague-Dawley rat fetuses at GD 19 (Fig. 1A–1D). Treatment with DBP from GD 12 to 19 at the maternal daily gavage dose of 500 mg/kg (but not at 10 mg/kg and 50 mg/kg) increased significantly (p < 0.05) the hepatic CYP 2B1 protein in both male and female fetuses (Fig. 1A and 1B). The level of hepatic 3A1 protein was not affected by in utero exposure to DBP in either male or female fetuses (Fig. 1C and 1D). A basal expression of CYP4A1 was also detected in fetal liver; this CYP isoform is significantly induced by DBP treatment in both female and male fetuses at the 500 mg/kg dose level (Fig. 1E and 1F). In several cases (female and male CYP2B1 and male CYP4A1), the amounts of enzyme proteins seemed to decrease from the control group to the 5 mg/kg and 50 mg/kg DBP groups before they became significantly induced at the 500 mg/kg level. These downward trends were not statistically significant.
Nuclear receptor CAR and PXR proteins were detectable in the livers of both male and female rat fetuses at GD 19 (Fig. 2). No difference in CAR protein level was observed in males or females between control and DBP-treated fetuses (Fig. 2A and 2B). The level of PXR protein in GD 19 fetal liver was increased in both male and female rats in the 500 mg/kg group (Fig. 2C and 2D), whereas the doses of 10 and 50 mg DBP/kg had no effect in this regard. The protein levels of hepatic PPAR and CYP4A were also increased in both male and female fetuses in the 500 mg/kg DBP group (data not shown).
The proteins of CAR, PXR, CYP 2B1, and CYP 3A1 were detected in liver samples of the pregnant dams (data not shown). The basal levels of these proteins in the maternal liver were greatly enhanced beyond those observed in the fetal liver. Di-n-butyl phthalate exposure did not cause an appreciable change in the levels of CAR and PXR proteins; however, the amounts of CYP2B1 and 3A1 were moderately increased in the 500 mg/kg dose group (data not shown).
Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) was performed to determine the amounts of mRNA of CAR, PXR, CYP 2B1, CYP 3A1, and the steroid-conjugating enzymes UGT2B1 and EST. Liver mRNA levels of both CYP 2B1 and 3A1 were increased twofold in the dams exposed to 500 mg DBP/kg compared to the controls (Fig. 3). In addition, mRNA levels of EST were increased twofold and threefold at the 50 and 500 mg/kg doses, respectively, whereas the CAR mRNA was increased fourfold at the 500 mg/kg dose level. In the male fetuses, hepatic CYP2B1 mRNA was markedly increased by the 500 mg/kg DBP treatment, which also increased the PXR mRNA (Fig. 3). Female fetuses showed a response pattern identical to that of male fetuses, with both CYP2B1 and PXR mRNA increases at similar dose–response magnitudes (data not shown). No treatment-related differences were detected in mRNA expression of CYP 3A1, UGT2B1, EST, or CAR in fetal liver.
The ability of DBP to activate PXR and CAR was investigated in cell lines transiently transfected with expression plasmids for the receptors and luciferase reporters. Constitutive androstane receptor activation was tested with rat CAR-transfected HepG2 cells and a dual luciferase reporter assay, whereas PXR activation was tested with PXR-transfected COS-7 cells and a dual luciferase reporter assay. Both systems have been used in numerous studies, and both were proven to be sensitive and specific assay tools for detecting the activation of the respective receptors (Wyde et al., 2003; Yoshinari et al., 2001; Zhang et al., 1999). In the CAR assay, we used androstenol (an inverse agonist) (Forman et al., 1998) to repress the constitutive CAR activity and 1,1-dichloro-2,2-bis (p-chlorophenyl)ethylene (DDE) as a positive control (Wyde et al., 2003). Di-n-butyl phthalate treatment did not result in CAR activation; rather, it caused slight inhibition of the CAR transcriptional activity (Fig. 4A). Cells treated with the inverse agonist androstenol (4 μM) demonstrated approximately an 80% inhibition in CAR transcriptional activity, and this inhibition was reversed by DBP treatment in a dose-dependent manner in the concentration range of 5 to 50 μM (Fig. 4B).
For PXR assays, we used pregnenolone 16--carbonitrile (PCN) and rifampicin as rat and human PXR positive controls, respectively (Zhang et al., 1999). The PXR transactivation assay demonstrated an ability of DBP to activate both rat and human PXR (Fig. 5). Activation of the rat PXR occurred at a lower dose (5 μM) than that for human PXR (20 μM). The peak activation of rat PXR occurred at 20 μM of DBP; that of human PXR, at 50 μM.
The ability of MBP, the main hydrolytic product of DBP, to activate PXR and CAR was also investigated. Mono-butyl-phthalate treatment did not increase the CAR receptor activity. nor did it reverse the repression of CAR by androstenol (Fig. 6A and 6B). Mono-butyl-phthalate treatment did, however, increase rat (but not human) PXR activation approximately twofold at the concentration of 50 μM (Fig 6C and 6D); this increase, however, was much smaller than the 8-fold induction caused by DBP (Fig. 5A).
DISCUSSION
At the fetal stage, the developing animal is particularly sensitive to reproductive effects of DBP, which can manifest at maternal doses that are without apparent effect on the dam (Mylchreest et al., 1998, 1999; Parks et al., 2000). In fetal testes, DBP exposure represses the genes involved in steroidogenesis, resulting in reduced androgen production (Lehmann et al., 2004; Mylchreest et al., 2002; Parks et al., 2000). The current study demonstrated that fetal liver, like fetal testis, is also susceptible to DBP through maternal exposure. We also demonstrated that DBP activates CAR and PXR, in addition to being a known activator of PPAR; these nuclear receptors are likely among the key regulators for DBP effects on liver enzymes.
The no-observable-adverse-effect level (NOAEL) of DBP-caused reproductive development effects in male rats was established at the 50 mg/kg maternal gavage dose (Mylchreest et al., 2000), whereas the lowest-observable-adverse-effect level (LOAEL) of DBP-caused inhibition on the expression of testicular steroidogenic genes was determined to be the 50 mg/kg maternal gavage dose (Lehmann et al., 2004). The current study detected DBP effects on the metabolic apparatus in the maternal and fetal liver at the 500 mg/kg dose level in proteins (CYP2B1 and PXR) and at the 50 mg/kg dose level in mRNA (CYP3A1, CYP2B1, and EST in the dams and CYP2B1 and PXR in the fetuses). The Lehmann et al. study (2004) demonstrated coordinated changes in gene expression of key testicular steroidogenic factors and testosterone production at the 50 mg/kg dose level. We do not know whether the similar sensitivities to DBP treatment of hepatic-metabolizing enzymes and testicular steroidogenic enzymes are based on shared mechanisms in the liver and testis.
Phthalates belong to a class of peroxisome proliferator chemicals. Exposure to those chemicals evokes a set of pleiotropic responses that include hepatocellular hypertrophy, hyperplasia, and induction of metabolic enzymes in rodent liver (Lock et al., 1989); these effects are known to be mediated mainly through PPAR (Reddy and Hashimoto, 2001). The ability of DBP to activate PPAR was demonstrated in an in vitro reporter gene transactivation assay (Lapinskas et al., 2005). This activation of PPAR explains a DBP-caused increase in hepatic CYP4A expression (Lapinskas et al., 2005), since CYP4A is a well-characterized target gene of PPAR (Lee et al., 1995; Ripp et al., 2002). As expected, the present study found that DBP exposure induced hepatic CYP4A1, and this induction, presumably mediated by PPAR, was operative at the fetal stage.
However, PPAR activation cannot explain the induction of CYP2B1 and 3A1 by DBP in the present study. In PPAR-null mouse, CYP3A11, a mouse homolog of the rat CYP3A1 gene, is inducible by xenobiotics (Ripp et al., 2002). In contrast, CYP2B was not inducible in CAR-null mouse, and CYP3A was not inducible in PXR-null mouse (Sonoda and Evans, 2003). Constitutive androstane receptor and PXR regulate hepatic genes, including the CYP2B and 3A families (Wang and Negishi, 2003; Wei et al., 2002). Thus, activation of PXR and CAR, but not PPAR, was likely required for the CYP3A1 and 2B1 induction in fetal rat liver by DBP. Indeed, DBP was shown to enhance the expression of hepatic CYP2B and 3A, whereas the PPAR agonist Wy-14,643 did not (Fan et al., 2004), further supporting that activating PXR and CAR, rather than PPAR, is responsible for the DPB effects. We have demonstrated in the present study that DBP interacts directly with CAR and PXR; such interactions are highly likely to be the mechanisms for DBP induction of genes in the CYP2B and 3A families (Wang and Negishi, 2003; Wei et al., 2002). The manner in which DBP activates PXR resembles the activation of PXR by DDE (Wyde et al., 2003). Di-n-butyl phthalate interacts differently with CAR than with PXR. Although DBP did not change the constitutive activity of CAR, it reversed the androstenol-imposed CAR repression; this type of CAR activation has been shown for other CAR activators as well (Blizard et al., 2001). Although we did not examine enzyme activities in these experiments, transcriptional increase of the hepatic CYP enzymes is known to correlate well with their protein levels and catalytic activities (Fan et al., 2004; Wyde et al., 2003; You et al., 1999).
We noted that, at the lower doses of DPB used in this study (5 mg/kg and 50 mg/kg), the protein amounts of several CYP isoforms seemed to be reduced, contrary to inductions at 500 mg/kg dose level. Similar reduction of CYP3A1 was previously reported to be associated with treatment with DDE and mifepristone (RU486), both PXR activators (Schuetz et al., 2000; Wyde et al., 2003). One potential mechanism for DBP to have such a seemingly biphasic effect on CYP expression is the mediation of glucocorticoid actions. The glucocorticoid receptor (GR) is essential for both basal and stimulated expression of CYP2B (Schuetz et al., 2000). Glucocorticoid-receptor–enhanced CYP expression is not mediated through cis-acting element but through complex protein–protein interactions (Honkakoski and Negishi, 2000). Activation of GR results in enhanced expression of PXR and RXR (Pascussi et al., 2000); the latter is the heterodimer partner of both PXR and CAR. Di-n-butyl phthalate may thus act either through an inhibition on steroidogenesis (reducing glucocorticoid level) or through displacement of endogenous activators at PXR (and possibly other CYP-regulating nuclear receptors) to cause reduction in certain CYP isoforms at specific doses.
Phthalic acid was reported to promote PXR interaction with steroid hormone receptor coactivator-1 (SRC-1), to increase PXR transcriptional activity in reporter gene assay, and to induce CYP3A1 in adult male rat liver (Masuyama et al., 2000). In the current study, we demonstrated that fetal liver is also susceptible to the effects of phthalates in regard to metabolic enzyme induction. We detected the proteins of CAR, PXR, AhR, and PPAR in fetal liver tissue. These receptors function as ligand-responsive transcriptional factors regulating hepatic induction of CYP2B1, 3A1, 1A1, and 4A1 in the rat. Constitutive androstane receptor and PXR also cross-regulate the CYP2B1 and 3A1 genes (Honkakoski et al., 2003). Detection of these receptor regulators of hepatic CYP enzymes at the fetal stage suggests physiological roles of these receptors in sensing and regulating the fetal environment. Although the CYP enzymes are regulated by ligand activation of these receptors, treatment-caused changes in receptor expression level may also play a role in controlling target gene expression. By examining the relationship between nuclear receptor expression and their target enzyme expression, we found no consistent coupling between increase in receptor expression and increase in enzyme expression.
A number of hepatocyte-enriched transcription factors are essential in coordinating gene expression during fetal liver differentiation (Cereghini, 1996); the differentiation process is necessary for the developing liver to acquire metabolic capacity. Hepatocyte nuclear factor-4 (HNF4) is a transcriptional coregulator of CAR and PXR for the human CYP3A4 gene (Tirona et al., 2003). In addition, HNF4 is a transcriptional factor for the PXR gene in fetal hepatocytes (Kamiya et al., 2003). In the present study, DBP treatment enhanced the expression of PXR and PPAR. Whether DBP interacts with hepatic transcriptional factors such as HNF4 remains to be seen; such interaction, if exists, would provide a mechanism for DBP-caused changes in the expression of PXR and PPAR.
Di-n-butyl phthalate toxicity is attributed in large part to MBP, a major hydrolysis product of DBP in vivo (Kavlock et al., 2002). In contrast to the ability of DBP to activate CAR and PXR, MBP showed little or no ability to activate these receptors. Similarly, MBP activation of PPAR was reported to be insignificant (Lapinskas et al., 2005). The fact that only very high doses of DBP caused induction of CYP enzymes suggests the possibility that a portion of the parent compound may escape the initial metabolism at high doses, that it may reach fetal liver cells, and that it may activate the corresponding receptors. Another possibility is that MBP may alter gene expression of CYP enzymes through mechanisms that are receptor independent.
In addition to CYP enzymes, CAR and PXR also regulate the expression of conjugating enzymes in the families of glutathione S-transferases (GST), UDP-glucuronosyltransferases (UGT), sulfotransferases (SULT), and multidrug-resistance–associated proteins (MRP) (Maglich et al., 2002; Xie et al., 2003). Although we detected only a slight change of UGT2B1 mRNA in dam liver (but not in fetuses), we found greater changes (over twofold and threefold at 50 and 500 mg/kg doses) for the expression of estrogen sulfotransferase. The induction of sulfotransferase is likely a consequence of activating CAR, instead of PXR, because the rat CAR activator TCPOBOP, but not the rat PXR activator PCN, was shown to be associated with regulating sulfotransferase expression (Maglich et al., 2002). The effects of phthalate on UGTs and SULTs are important, because the conjugating reactions catalyzed by these enzymes render the many endogenous molecules and chemical metabolites highly hydrophilic and easily excreted from the body (You, 2004). The lack of response in conjugating enzyme expression to DBP in the fetal liver suggests that a change in these conjugation pathways is not a significant factor in regulating hormone activities in the fetuses.
Although there may be implications with regard to developmental dysregulation, the significance of fetal liver effects caused by DBP after maternal exposure remains to be adequately appraised. The diverse nature of DBP interaction with nuclear receptors PXR, CAR, and PPAR, among possible others, and the extensive involvement of these nuclear receptors in regulating numerous metabolic pathways suggest broad potentials of DBP modulation on the metabolism of lipids, steroids, and other biological processes, including lipid homeostasis, cholesterol metabolism, and steroidogenesis in the gonads.
ACKNOWLEDGMENTS
This research was funded by the American Chemistry Council Long-Range Research Initiative (to L.Y.) and a National Institutes of Health grant (R01-GM61988) (to B.Y.). We thank Drs. Susan Borghoff and Kamin Johnson for reading the manuscript and Dr. Barbara Kuyper for editorial review. Conflict of interest: none declared.
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