Di-n-Butyl Phthalate Activates Constitutive Androstane Receptor and
Pregnane X Receptor and Enhances the Expression of Steroid-
Metabolizing Enzymes in the Liver of Rat Fetuses
Michael E. Wyde,* Shaun E. Kirwan,* Fan Zhang,* Ashley Laughter,* Holly B. Hoffman,* Erika Bartolucci-Page,*
Kevin W. Gaido,* Bingfang Yan,† and Li You*,1
*CIIT Centers for Health Research, Research Triangle Park, North Carolina; †Department of Biomedical Sciences,
University of Rhode Island, Kingston, Rhode Island
Received March 17, 2005; accepted May 9, 2005
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
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
foranalyses. IncreasedproteinandmRNAlevelsofCYP 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 sulfotransfer-
ase and UDP-glucuronosyltransferase 2B1 in the dams but not in
the fetuses. Xenobiotic induction of CYP3A1 and 2B1 is known to
bemediated by thenuclearhormone receptors pregnane Xreceptor
(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;
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 Examina-
the general U.S. population, including children and women of
child-bearing age (CDC, 2003); however, the estimated expo-
sure levelsarewell under theEnvironmental Protection Agency
reference dose (RfD) of 0.1 mg/kg/day (USEPA, 2005).
Di-n-butyl phthalate has been demonstrated to be a reproduc-
tive 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 ab-
normal 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 pro-
duction 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 (PPARa, b, c) may also repre-
sent a mechanism for the phthalate-causedreproductive toxicity;
but the evidence in this regard is not yet conclusive (Corton and
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 PPARa (Lapinskas et al., 2005) and
causes changes in the expression of a number of PPARa-
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 PPARa and
PPARc (Hurst and Waxman, 2003; Lapinskas et al., 2005). The
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Research, 6 Davis Drive, Research Triangle Park, NC 27709–2137. Fax: (919)
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TOXICOLOGICAL SCIENCES 86(2), 281–290 (2005)
Advance Access publication May 18, 2005
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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
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-metabo-
lizing enzymes to DBP exposure and to establish relevant
mechanisms. We evaluated the expression levels in fetal liver
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 nu-
clear receptors CAR and PXR. Such enzyme modulations sug-
gest a potential for DBP to interfere with steroid and lipid
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
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
cornoil vehicle from GD 12 to GD 19.Di-n-butyl phthalatewasadministeredat
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 PPARa. 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 tissuewere denatured and separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12%
polyacrylamide. Proteins were transferred to nitrocellulose membranes; trans-
fer 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 PPARa. After
incubation with primary antibody, membranes were incubated with horseradish
peroxidase–linked anti-rabbit (CYP3A1, PXR, PPARa, and CAR) or anti-goat
(CYP1A1, CYP2B1, and CYP 4A1) IgG secondary antibodies and visualized
on film exposed to enhanced chemiluminescence (Hyperfilm-ECL, Amer-
sham). 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-PPARa 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 back-
ground 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 Tm¼
60?C, high Tm¼ 64?C, optimum Tm¼ 62?, amplicon length ¼ 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 electropho-
resis 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
WYDE ET AL.
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