Endocrine Disrupting Chemicals, Phthalic Acid and Nonylphenol, Activate Pregnane X Receptor-Mediated Transcription
Hisashi Masuyama,
Yuji Hiramatsu,
Mamoru Kunitomi,
Takafumi Kudo and
Paul N. MacDonald
Department of Obstetrics and Gynecology (H.M., Y.H., M.K.,
T.K.) Okayama University Medical School Okayama, 700-8558,
Japan
Department of Pharmacology (P.N.M.) Case Western
Reserve University Cleveland, Ohio 44106
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ABSTRACT
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Recently, Pregnane X receptor (PXR), a new member
of the nuclear receptor superfamily, was shown to mediate the effects
of several steroid hormones, such as progesterone, glucocorticoid,
pregnenolone, and xenobiotics on cytochrome P450 3A genes (CYP3A)
through the specific DNA sequence for CYP3A, suggesting that
PXR may play a role in steroid hormone metabolism. In this
paper, we demonstrated that phthalic acid and nonylphenol,
endocrine-disrupting chemicals (EDCs), stimulated PXR-mediated
transcription at concentrations comparable to those at which they
activate estrogen receptor-mediated transcription using a transient
reporter gene expression assay in COS-7 cells. However, bisphenol A,
another EDC, had no effect on PXR-mediated transcription, although this
chemical significantly enhanced ER-mediated transcription. In the yeast
two-hybrid protein interaction assay, PXR interacted with two nuclear
receptor coactivator proteins, steroid hormone receptor coactivator-1
and receptor interacting protein 140, in the presence of
phthalic acid or nonylphenol. Thus, EDC-occupied PXR may regulate its
specific gene expression through the receptor-coactivator interaction.
In contrast, these EDCs had no effect on the interaction between PXR
and suppressor for gal 1, a component of proteasome. Finally, the
expression of CYP3A1 mRNA in the liver of rats exposed to phthalic acid
or nonylphenol markedly increased compared with that in rats treated
with estradiol, bisphenol A, or ethanol as assessed by competitive
RT-PCR. These data suggest that EDCs may affect endocrine functions by
altering steroid hormone metabolism through PXR.
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INTRODUCTION
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The nuclear receptor superfamily consists of more than 150
different proteins that have evolved to mediate a complex array of
extracellular signals into transcriptional responses. There are many
ligands for these nuclear receptors, including steroid
hormones, such as estradiol (E2), progesterone
(P), and glucocorticoid, and nonsteroid hormones, such as vitamin D,
retinoids, thyroid hormone, and prostanoids. These receptors form
homodimers or heterodimers with RXR and directly associate with
specific DNA sequences known as hormone-responsive elements located in
the promoters of specific genes (1, 2, 3). The DNA-receptor complex
interacts with basal transcriptional machinery and nuclear receptor
coactivator proteins, resulting in ligand-dependent induction of
transcription (3, 4, 5). Recently, pregnane X receptor (PXR), a new member
of the nuclear receptor superfamily, has been shown to mediate the
effects of several steroid hormones, such as P,
pregnenolone, glucocorticoid, synthetic glucocorticoids,
antiglucocorticoids, and xenobiotics on the cytochrome P450 3A genes
(CYP3A) in the mouse, rat, and man (6, 7, 8, 9, 10, 11, 12). Like nonsteroid hormone
receptors, PXR binds as a heterodimer with retinoid X receptor to
specific DNA sequences, including those upstream of CYP3A, and
regulates expression of target genes (6, 7, 8, 11).
The cytochrome P450 family consists of heme-containing monooxygenases
that function in the oxidative metabolism of a wide variety of
endogenous substances and xenobiotics. Specifically, the CYP3A
subfamily is involved in the metabolism of endogenous substrates such
as steroids, bile acids, and retinoic acid. In addition, this subfamily
also plays important roles in the metabolism of procarcinogen and
pharmaceutical agents, including innumerable drugs, chemical
carcinogens, mutagens, and other environmental contaminants (13, 14).
The fact that PXR has been shown to be activated by several steroids
and other exogenous compounds that are known to induce the CYP3A genes
(6, 7, 8) suggests a novel endocrine signaling pathway that regulates the
metabolism of steroids and xenobiotics through PXR.
Some environmental agents have been shown to disrupt the endocrine
functions in many species through a variety of pathways including the
change of steroidogenesis (15). Nonylphenol, one of these environmental
agents, has been demonstrated to induce CYP3A expression (16). Thus, we
examined whether some endocrine-disrupting chemicals (EDCs) including
nonylphenol activate the PXR-mediated transcription through the CYP3A1
motif in the transient reporter assay. We also checked whether PXR
interacts with two nuclear receptor coactivators, steroid
hormone receptor coactivator-1 (SRC-1) (17) and receptor interacting
protein 140 (RIP140) (18), in the presence of EDCs. Finally,
the effect of EDCs on CYP3A1 mRNA expression in the liver of
EDC-treated rats was analyzed using competitive RT-PCR. The
results of these experiments provide some evidence that EDCs stimulate
PXR-mediated transcription through the interaction between PXR and
nuclear receptor coactivator proteins, suggesting that EDCs may affect
the endocrine functions by altering the steroid hormone metabolism
through PXR.
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RESULTS
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EDCs Stimulate PXR-Mediated Transcription
A transient reporter expression assay was performed in COS-7 cells
to examine whether EDCs enhance PXR-mediated transcription.
Interestingly, phthalic acid and nonylphenol enhanced PXR-mediated
transcription, and this effect was almost equal to the
transcriptional level of the cells treated with P, one of the natural
steroids that activate PXR (6). In contrast, E2,
dithiothreitol (DDT), and bisphenol A had no effects on the
transcription (Fig. 1A
). These effects
were dependent on the concentration of ligands and significantly
increased at 10 nM, but bisphenol A had no effect
on PXR-mediated transcription at any of the concentrations tested (Fig. 1B
). These data suggest that phthalic acid and nonylphenol are
exogenous ligands for PXR. We also determined the concentration of EDCs
that enhanced ER-mediated transcription in this assay. Bisphenol A,
phthalic acid, and nonylphenol had similar positive effects comparable
to those of E2 on ER-mediated transcription (Fig. 1C
). However, these EDCs had significant effects only at high
concentrations, while E2 significantly stimulated
the transcription at 10 pM (Fig. 1D
).

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Figure 1. EDCs Enhanced PXR-Mediated Transcription
A, COS-7 cells were transfected with 1 µg of
(CYP3A1)2-tk-CAT reporter gene construct together with 0.5
µg of the PXR expression plasmid or empty vector (pSG5). The cells
were treated with 10-6 M of
estradiol, progesterone, DDT, bisphenol A, phthalic
acid, nonylphenol, or ethanol vehicle for 36 h. CAT activity was
quantified using an ELISA kit. The results represent the mean ±
SD of triplicate determinations. B, COS-7 cells were
transfected with 1 µg of (CYP3A1)2-tk-CAT reporter gene
construct together with 0.5 µg of the PXR expression plasmid. The
cells were treated with increasing concentrations of progesterone,
phthalic acid, nonylphenol, or bisphenol A for 36 h. CAT activity
was quantified using an ELISA kit. The results represent the mean
± SD of triplicate determinations. Students
t test was used to determine whether treated values were
significantly different from the control with P <
0.05 as the limit of significance (*, P < 0.01,
**, P < 0.05). C, COS-7 cells were
transfected with 1 µg of (ERE)2-G-CAT reporter gene
construct together with 0.5 µg of the ER expression plasmid or
empty vector (pSG5). The cells were treated with 10-6
M of estradiol, DDT, bisphenol A, phthalic acid,
nonylphenol, or ethanol vehicle for 36 h. CAT activity was
quantified using ELISA kit. The results represent the mean ±
SD of triplicate determinations. D, COS-7 cells
were transfected with 1 µg of (ERE)2-G-CAT reporter gene
construct together with 0.5 µg of the ER expression plasmid. The
cells were treated with increasing concentrations of estradiol,
phthalic acid, or nonylphenol for 36 h. CAT activity was
quantified using an ELISA kit. The results represent the mean ±
SD of triplicate determinations. Students
t test was used to determine whether treated values were
significantly different from the control with P <
0.05 as the limit of significance (*, P < 0.01,
**, P < 0.05).
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Effect of EDCs on the Interaction between PXR and Coactivator
Proteins
A two-hybrid protein interaction assay was used to examine whether
PXR interacted with coactivator proteins, which are very important for
the nuclear receptor-mediated transcription (3, 4, 5), in the presence of
EDCs. As illustrated in Fig. 2
, A and B,
PXR interacted with two nuclear receptor coactivators, SRC-1 and
RIP140, in the presence of P, phthalic acid or nonylphenol, which
stimulated PXR-mediated transcription (Fig. 1A
). However, bisphenol A
and dichlorodiphenyltrichloroethane (DDT), which had no effect on
PXR-mediated transcription, did not affect this interaction. In
contrast, EDCs had no effect on the interaction between PXR and
suppressor for gal-1 (SUG1), a component of proteasome (19, 20) (Fig. 2C
). We also examined the effects of these EDCs on the interaction of
another steroid hormone receptor with SRC-1. As shown in Fig. 2D
, none
of the EDCs tested here affected the interaction between vitamin D
receptor (VDR) and SRC-1, suggesting that the effects of EDCs are
specific for the interactions between PXR and coactivators (Fig. 2D
).
The effect on the PXR interaction with SRC-1 was dependent on the
concentration of ligands and significantly increased at 10
nM, a concentration comparable to those that activated
PXR-mediated transcription (Fig. 2E
).

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Figure 2. The Effect of EDCs on the Interaction between PXR
and Coactivator Proteins
A, Yeast expressing the pAS1-PXR or empty vector (pAS1) and
pAD-SRC-1 two hybrid plasmids were grown for 24 h at 30 C in the
presence of 10-6 M progesterone, DDT,
bisphenol A, phthalic acid, nonylphenol, or ethanol vehicle. PXR-SRC-1
interaction was assessed in a ß-galactosidase assay. The results
represent the mean ± SD of triplicate independent
cultures. B, Yeast expressing the pAS1-PXR or pAS1 and pAD-RIP140
two-hybrid plasmids were grown for 24 h at 30 C in the presence of
10-6 M of progesterone, DDT, bisphenol A,
phthalic acid, or nonylphenol, or ethanol vehicle. PXR-RIP140
interaction was assessed in a ß-galactosidase assay. The results
represent the mean ± SD of triplicate independent
cultures. C, Yeast expressing the pAS1-PXR or pAS1 and pAD-SUG1
two hybrid plasmids were grown for 24 h at 30 C in the presence of
10-6 M of progesterone, DDT, bisphenol A,
phthalic acid, or nonylphenol, or ethanol vehicle. PXR-SUG1 interaction
was assessed in a ß-galactosidase assay. The results represent the
mean ± SD of triplicate independent cultures. D,
Yeast expressing the pAS1-VDR or empty vector (pAS1) and pAD-SRC-1
two-hybrid plasmids were grown for 24 h at 30 C in the presence of
10-6 M progesterone, DDT, bisphenol A,
phthalic acid, nonylphenol, 1,25-dihydroxyvitamin D3, or
ethanol vehicle. VDR-SRC-1 interaction was assessed in a
ß-galactosidase assay. The results represent the mean ±
SD of triplicate independent cultures. E, Yeast expressing
the pAS1-PXR and pAD-SRC-1 two-hybrid plasmids were grown for 24 h
at 30 C with increasing concentrations of progesterone, phthalic acid,
or nonylphenol. PXR-SRC-1 interaction was assessed in a
ß-galactosidase assay. The results represent the mean ±
SD of triplicate independent cultures. Students
t test was used to determine whether treated values were
significantly different from the control with P <
0.05 as the limit of significance (*, P < 0.01).
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Effect of Phthalic Acid on the Expression of CYP 3A1 in the
Rat Liver
The expression of CYP3A1 and ß-actin mRNA in the livers of rats
after 24 h exposure to several chemicals was analyzed using the
competitive RT-PCR method. The expression of CYP3A1 mRNA increased
markedly in rats exposed to phthalic acid, nonylphenol, or P, relative
to the expression in rats treated with E2,
bisphenol A, or ethanol (Fig. 3
, A and
B). Treatment with E2 moderately enhanced the
expression of CYP3A1 mRNA, and bisphenol A had a weak effect on the
expression of CYP3A1 mRNA. The housekeeping gene, ß-actin,
was used to determine the constitutive level of gene transcription and
to control for variations in RNA recoveries from each liver
specimen.

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Figure 3. The Effect of EDCs on the Expression of CYP3A1 mRNA
A, The total RNA was isolated from livers of rat exposed to phthalic
acid, nonylphenol, bisphenol A, estradiol, progesterone, or ethanol and
analyzed for the mRNA expression of CYP3A1 gene and ß-actin using
competitive RT-PCR. The PCR products were separated on 3% Nu-Sieve
agarose gels and visualized by ethidium bromide. B, The band
intensities were densitometrically measured and quantified using Image
Scanner T-9500 and Bio Image software. The data are averages of two
determinations of mRNA from two rats. Students t test
was used to determine whether treated values were significantly
different from the control with P < 0.05 as the
limit of significance (*, P < 0.01).
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DISCUSSION
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The endocrine disrupting chemical (EDC) has been defined as
an exogenous agent that interferes with the synthesis, secretion,
transport, binding, action, or elimination of natural hormones in the
body, which are responsible for the maintenance of homeostasis,
reproduction, development, and/or behavior (21). These chemicals can
alter endocrine functions through a variety of mechanisms,
including steroid hormone receptor-mediated changes in protein
synthesis, interference with membrane receptor binding,
steroidogenesis, or synthesis of other hormones (15). Major
chemicals, such as phthalates, alkylphenols, bisphenol A, and DDT, have
been shown to disrupt estrogenic actions mainly through the binding to
estrogen or androgen receptors (15). However, other potential cellular
mechanisms have not been vigorously explored. Here, we demonstrate that
a kind of EDCs enhanced PXR-mediated transcription at concentrations
comparable to those at which they activate ER-mediated transcription
and that the expression of endogenous CYP3A1 mRNA increased by
treatment with these EDCs. Other EDCs, nonplanar polychlorinated
biphenyls, are known to up-regulate CYP3A expressions, and these
compounds have been shown to activate PXR-mediated transcription at
concentrations comparable to those at which phthalic acid and
nonylphenol activated PXR-mediated transcription in the present
experiments (12). Since the CYP3A family, which has been shown to be
one of the specific genes regulated by PXR (6, 7, 8), hydroxylates
endogenous steroids, including cortisol, progesterone, and testosterone
(13, 14), it is possible that these EDCs might have some effects on
endocrine function by altering steroid hormone metabolism through PXR
as well as by the binding to estrogen receptor. This speculation is
supported by the fact that rifampicin, a PXR ligand in humans (7), has
been shown to affect plasma levels of estradiol and the
pharmacokinetics of oral contraception (22, 23). We here demonstrated
that, although E2 and bisphenol A had no effect
on PXR-mediated transcription in our experiments, both induced moderate
or weak CYP3A gene expression in the treated rats. In addition,
E2 has been shown to up-regulate the expression
of other CYP3A family members (24). Further experiments will be needed
to determine the mechanisms by which E2 induces
CYP3A expression.
It is becoming increasingly clear that protein-protein contacts between
the receptor and the basal transcriptional machinery are important for
ligand-mediated transactivation or repression by nuclear receptors.
Nuclear receptors directly contact several general transcription
factors (GTFs) in the preinitiation complex (PIC). The interaction of
receptors with these GTFs is thought to either recruit these limiting
factors to PIC assembly or to stabilize the PIC itself (2). Moreover,
coactivator proteins, including SRC-1 (17), estrogen
receptor-associated protein (ERAP 160) (25), and RIP140 (18), interact
in a ligand-dependent manner with several members of the nuclear
receptor superfamily to enhance ligand-induced transactivation (3, 4, 26). In this paper, we showed that PXR interacted with the coactivator
proteins SRC-1 and RIP140 in a ligand-dependent manner similar to that
of other nuclear receptors. In addition, we demonstrated that phthalic
acid and nonylphenol enhanced the interaction between PXR and these
coactivator proteins. These data suggest that phthalic acid and
nonylphenol enhanced PXR-mediated transcription through the interaction
of PXR with coactivators. Importantly, these compounds did not affect
the interaction between PXR and SUG1. SUG1 has been described as a
component of proteasome (20), which is an enzyme complex responsible
for major protein degradation (27, 28, 29). We have also reported that SUG1
plays some roles in nuclear receptor degradation by proteasome (30).
PXR does not interact with SUG1 in the presence of EDCs, although
progesterone enhanced the interaction between PXR and SUG1, suggesting
that EDC-occupied PXR might have a conformational change distinct from
that of the natural steroid-occupied receptor. The crystallization of
the ligand-binding domain of the PXR with progesterone and EDCs should
provide important insights into the conformational changes that occur
in the liganded receptor. Since PXR interacts with SUG1 in the presence
of natural steroids, but does not interact in the presence of phthalic
acid or nonylphenol, the EDCs-occupied PXR may be more slowly degraded
than the steroid hormone-occupied receptor, which might result in the
difference of the PXR-mediated gene expression. Additional studies are
ongoing to investigate this question.
In summary, we demonstrated that some EDCs enhanced PXR-mediated
transcription through the PXR interaction with coactivator proteins.
Also, these EDCs had more positive effects on the expression
of CYP3A1 mRNA, which is a target gene through PXR and plays important
roles in the metabolism of steroid hormones and exogenous substrates,
than did vehicle, other EDCs, or estradiol. These data suggest that
EDCs may affect the endocrine functions by altering the steroid hormone
metabolism through PXR.
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MATERIALS AND METHODS
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Materials
Isopropylidenediphenol (bisphenol A), phthalic acid bis
(2-ethylhexel ester) (phthalic acid), P, and E2
were purchased from Sigma (St. Louis, MO).
1,25-Dihydroxyvitamin D3 was kindly provided by
Dr. M. R. Uskokovic. 4-Nonylphenol (nonylphenol), which is a
mixture of compounds with branched side chains, and DDT were obtained
from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Male Wistar rats,
200300 g, bred in our laboratory, were used in these studies. Food
and water were available ad libitum. All materials for
competitive RT-PCR were purchased from TAKARA Co., Ltd. (Kyoto,
Japan).
Transient Transfection Studies
COS-7 cells were cultured in DMEM medium without phenol
red supplemented with 10% charcoal-stripped calf serum. The
(CYP3A1)2-tk-CAT containing two copies of the
CYP3A1 motif, which is a direct repeat of the nonsteroid nuclear
receptor half-site sequence AGTTCA separated by a three-nucleotide
spacer (31, 32), and pSG5-PXR expression plasmid containing full-length
mouse PXR cDNA were obtained from Dr. S. A. Kliewer (6). COS-7
cells were cotransfected with 1 µg of reporter gene construct
[CYP3A1)2-tk-CAT or
(ERE)2-G-CAT] and 0.5 µg of receptor
expression vector (pSG5-PXR or pSG5-ER
) or empty vector (pSG5). In
all transfections, liposome-mediated transfections were accomplished
with lipofectamine (Life Technologies, Inc., Gaithersburg,
MD) according to the manufacturers protocol. Transfected cells were
treated with either vehicle alone or the indicated concentrations of
steroid hormones or EDCs for 36 h. Cell extracts were prepared and
assayed for CAT (chloramphenicol acetyltransferase) activity. The
amount of CAT was determined with a CAT enzyme-linked immunosorbent
assay (ELISA) kit (5 Prime
3 Prime, Inc., Boulder, CO) according to
the manufacturers protocol.
Preparation of Two-Hybrid Expression Vectors and
ß-Galactosidase Assays
All two-hybrid plasmid constructs used the pAS1 (33) and
pAD-GAL4 (Stratagene, La Jolla, CA) yeast expression
vectors. The pAD-GAL4-SUG1, -SRC-1, and -RIP140 were previously
described (26). The full-length PXR was subcloned into the pAS1 to
examine the interaction with coactivator proteins in the two-hybrid
assay. The pAS1-PXR, -VDR (34), or empty vector (pAS1) was
cotransformed with pAD-GAL4-SUG1, -SRC-1, or -RIP140 into the yeast
strain Hf7c, which was made competent with lithium acetate.
Transformants were plated on media lacking leucine and tryptophan
(SC-leu-trp) and were grown for 4 days at 30 C to select for yeast that
had acquired both plasmids. Triplicate independent colonies from each
plate were grown overnight in 2 ml of SC-leu-trp with or without the
indicated concentrations of steroids or EDCs. The cells were harvested
and assayed for ß-galactosidase activity as described previously
(35).
Administration of Chemicals and Tissue Collection
The animals were administered phthalic acid, nonylphenol,
bisphenol A, E2, P (0.3 mg/kg), or ethanol via
intraperitoneal injection. Twenty-four hours after the injection, the
animals were killed under ether anesthesia, and the livers were
removed, immediately frozen, and stored at -70 C. The frozen tissue
was homogenized in a Polytron homogenizer, and total RNA was extracted
using the guanidine iodothiocyanate method (Trizol; Life Technologies, Inc.) according to the manufacturers
instructions.
RT-PCR
Each sample was treated with DNase I to remove genomic DNA
contamination. To confirm the absence of genomic DNA in the RNA
samples, PCR was performed directly on each RNA sample using primers
for CYP3A1 and ß-actin, and no PCR products were detected under this
condition. According to the protocol of the RNA PCR kit, 0.1 µg of
total RNA was reverse transcribed at 42 C for 20 min in 20 µl of
reaction solution containing 1xPCR buffer, 5 mM
MgCl2, 1 mM deoxynucleoside
triphosphates, 2.5 µM random 9 mers primer, 10 U
ribonuclease inhibitor, and 5 U AMV reverse transcriptase. The primers
for CYP3A1 (36) and ß-actin were as follows: CYP3A1 sense:
5'-ATCCGATATGGAGATCAC-3',3'; antisense: 5'-GAAGAAGTCCTTGTCTGC-3',
ß-actin sense: 5'-GTTTGAGACCTTCAACACCC-3'; 3' antisense:
5'-CTTGATCTTCATGGTGCTAG-3'. Each PCR sample contained 1xPCR buffer, 2
mM MgCl2, 10 pmol of primer mix for
CYP3A1 or ß-actin, and 1.25 U TAKARA LA Taq. Amplification
was carried out on a TAKARA PCR thermocycler with initial denaturation
at 94 C for 2 min, followed by 24 cycles of 94 C for 30 sec, 56 C for
30 sec, 72 C for 30 sec, and a final extension at 72 C for 2 min. The
number of PCR cycles resulting in PCR products in the linear
logarithmic phase of the amplification curve was determined. PCR
samples were electrophoresed on 3% Nu-Sieve agarose gel and visualized
by ethidium bromide. The amount of each electrophorectically separated
cDNA was quantitated densitometrically using an Image Scanner GT-9500
(Epson, Suwa, Japan) and Bio Image BQ 2.0 software (Bio Image, Ann
Arbor, MI).
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ACKNOWLEDGMENTS
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The authors thank Dr. Steven A Kliewer for providing mouse PXR.1
expression vector and CYP3A1 reporter vector.
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FOOTNOTES
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Address requests for reprints to: Hisashi Masuyama, M.D., Ph.D., Department of Obstetrics and Gynecology, Okayama University Medical School, 25-1, Shikata, Okayama, 700-8558, Japan.
Received for publication June 15, 1999.
Revision received November 23, 1999.
Accepted for publication December 1, 1999.
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