Estrogen-dependent gene regulation by an oxidative metabolite of diethylstilbestrol, diethylstilbestrol4',4'-quinone

Article abstract of DOI:10.1016/S0039-128X(97)00158-X

Diethylstilbestrol (DES) is a well-characterized carcinogen in humans and animals although its mechanisms of carcinogenicity are not yet known. While the estrogenic activity of DES is important, there is evidence that oxidative metabolism also plays an im

Full text of DOI:10.1016/S0039-128X(97)00158-X

Estrogen-dependent gene regulation by
an oxidative metabolite of
diethylstilbestrol, diethylstilbestrol-
4؅,4؆-quinone
Kun Chae,* Jonathan Lindzey,* John A. McLachlan,† and Kenneth S. Korach*
*Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National
Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA
and †Tulane-Xavier Center for Bioenvironmental Research, New Orleans, Louisiana, USA
Diethylstilbestrol (DES) is a well-characterized carcinogen in humans and animals although its mechanisms of
carcinogenicity are not yet known. While the estrogenic activity of DES is important, there is evidence that
oxidative metabolism also plays an important role for its toxicity. DES is oxidatively metabolized in vivo and in
vitro to a number of compounds including diethylstilbestrol-4Ј,4Љ-quinone (DQ), an unstable and reactive
intermediate, and Z,Z-dienestrol (ZZ-DIEN). Estrogen receptor (ER) binding assays with mouse uterine cytosol
indicate that DES, DQ and ZZ-DIEN have relative binding affinities of 286, 3.6 and 0.3, respectively, relative to
estradiol as 100. In addition, DQ binds irreversibly and specifically to ER suggesting that DQ may be biologically
active despite its rapid metabolism and lower binding affinity compared to DES. To test this, COS-1 cells were
transfected with an estrogen responsive reporter construct containing a VitA2 estrogen response element (ERE)
with or without an ER expression vector. In the presence of ER, treatments with DES, DQ and ZZ-DIEN resulted
in 11, 10, and 2-fold induction of chloramphenicol acetyltransferase (CAT) activity, respectively. This induction
was mediated by estrogen receptor since it was suppressed by pretreatment with a 10-fold excess of the pure
antiestrogen ICI 182,780. These data indicate that DQ is a biologically active intermediate that is capable of
transactivation of estrogen responsive genes through the ER. Furthermore, the data suggest that the ability of DQ
to irreversibly bind ER may result in persistent stimulation of ER. This persistent stimulation may be related to
the carcinogenicity of DES. (Steroids 63:149–157, 1998) © 1998 by Elsevier Science Inc.
Keywords: estrogen receptor; diethylstilbestrol; DES-quinone; transactivation
may play an important role in the estrogenic activity, car-
cinogenicity, or toxicity of DES.7,8 Indeed, it has been
reported that DQ covalently binds to peptides, proteins9 and
to DNA.10,11 Such a covalent DQ-ER complex might be
expected to produce sustained biological activity. In order
to investigate whether binding of DQ to ER might contrib-
ute to the estrogenic and carcinogenic effects of DES, we
compared the ability of DES and DQ to bind ER and
activate ER dependent gene activity in transient transfec-
tions.
Introduction
The synthetic estrogen, diethylstilbestrol (DES) has potent
estrogenic biological activity,1 and is a known carcinogen in
humans and in laboratory animals.2,3 The most significant
health effects of DES are due to in utero exposure that leads
to increased incidences of vaginal and cervical clear cell
adenocarcinoma in the adult female.2,3 Although the mech-
anism of the carcinogenesis of DES is not fully known, it is
possible that metabolites of DES play a role in its biological
activity.1,4 DES is metabolized to a number of reactive
metabolites including its oxidative metabolite, diethylstil-
bestrol-4Ј,4Љ-quinone (DQ), and further metabolized to a
non-carcinogenic metabolite Z,Z-dienestrol (ZZ-DIEN).5–7
The interaction of either DES or its metabolites with ER
Experimental
Materials
17␤-[2,4,6,7-3H]Estradiol (E2), 110 Ci/mmol, with Ͼ98% radio-
chemical purity was obtained from Du Pont-New England Nuclear
(Boston, Massachusetts). Unlabeled ZZ-DIEN and [3H]diethylstil-
bestrol (DES) with a specific activity of 46.5 Ci/mmol were
prepared by Chemsyn Science Laboratories (Lenexa, Kansas,
USA). ICI 182,780 was from ICI Pharmaceuticals (Macclesfield,
Address reprint requests to Kun Chae, Lab of Reproductive and Develop-
mental Toxicology NIEHS Receptor Biology Section, PO Box 12233,
101 TW Alexander Drive, Research Triangle Park, NC 27709, USA.
Received September 12, 1997; accepted November 20, 1997.
Steroids 63:149–157, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
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PII S0039-128X(97)00158-X

Papers
England, United Kingdom). Activated manganese (IV) oxide was
obtained from Aldrich Chemical Co. (Milwaukee, Wisconsin,
USA). Unlabeled diethylstilbestrol was obtained from Sigma (St.
Louis, Missouri, USA). HPLC-grade solvents were purchased
from Fisher (Fairlawn, New Jersey, USA). Fetal calf serum was
obtained from Life Technologies, Gibco BRL (Gaithersburg,
Maryland, USA).
male CD-1 [ICR] BR mice from Charles River Breeding Labora-
tories (Wilmington, Massachusetts, USA) were kept in
a
controlled environment with 12 h of light and 12 h of dark. Feed
and water were given ad lib. After sacrifice by cervical dislocation
at eight to ten weeks of age, the uteri were removed and frozen on
dry ice. The frozen uteri were then placed in ice-cold TEGM buffer
(10 mM Tris, 1.5 mM EDTA, 10% glycerol, 3 mM MgCl2, pH 7.6)
containing protease inhibitors (50 ␮g/mL each of leupeptin, soy-
bean trypsin inhibitor, antipapain, and chymostatin), and homog-
enized with a Polytron (Brinkmann Instrument, Westbury, New
York, USA) for 15 s at a speed setting of 6.5 at a ratio of 75:1 (mg
tissue weight/mL buffer). The homogenate was filtered through
100–125 ␮m mesh Nitex, centrifuged at 1,000 ϫ g for 10 min, and
then the supernatant was decanted and centrifuged at 45,000 rpm
for 50 min. The 105,000 ϫ g supernatant was used for cytosol
receptor binding assays.
High-performance liquid chromatography and
ultraviolet spectra
High-performance liquid chromatography (HPLC) was performed
on a Beckman (Fullerton, California) HPLC system with System
Gold software equipped with a Beckman 126 pump and 168 Diode
Array Detector monitoring at 254 and 280 nm. The C-18 reversed
phase column (IBM 4.6 ϫ 250 mm) was eluted with a mixture of
acetonitrile and water. The solvent was programmed from 50%
acetonitrile in water to 100% acetonitrile in 30 min at a flow rate
of 1 ml/min. Radioactivity was measured using a Beckman 171
Radioisotope Detector.
The stability of DQ in sFCS DMEM media containing COS-1
cells was measured by adding DQ (0.1 mM) to a mixture of COS-1
cells in DMEM media with 10% sFCS at 37°C. Ultraviolet spectra
scans were recorded on a Beckman DU-7 Spectrophotometer at the
indicated time intervals.
Aliquots (100 ␮L) of cytosol were incubated with 5 nM [3H]E2
and increasing concentrations of unlabeled competitors (0.1 nM–
100 nM). The mixtures were incubated at 4°C for 18 h, and 250 ␮L
of 60% hydroxylapatite (HAP) in TEGM buffer was added to each
tube to adsorb the bound ER. Tubes were then centrifuged at 1,000
ϫ g for 10 min, and the resulting HAP pellet was washed twice
with 3 mL of TEGM buffer and suspended in Ecoscint O scintil-
lation cocktail (National Diagnostics, Atlanta, Georgia). The ra-
dioactivity was measured on a Beckman LS 9800 scintillation
counter. Specific binding was determined as the difference be-
tween total (radioactive ligand only) and non-specific binding
(radioactive ligand with excess unlabeled ligand) in the prepara-
tion. The Kd (dissociation constants) values were determined using
Ligand Competition Analysis Software by Lundon Software (Cha-
grin Falls, Ohio). Relative binding affinities (RBA) were calcu-
lated from the competitive binding assay, with the binding affinity
for estradiol-17␤ set to 100.
Synthesis of DQ and [3H]DQ
DQ was synthesized according to the method described by Abul-
Hajj12 with modification. A mixture of diethylstilbestrol (2.7 mg)
and manganese (IV) oxide (MnO2) (0.87 mg) in 1 mL of chloro-
form was vortexed in an amber vial at room temperature for 5 min.
After removing the manganese oxide by centrifugation, the DQ in
chloroform solution was kept in ice and used for the experiments.
The DQ in chloroform is stable for up to a week when stored in the
dark at Ϫ20°C. [3H]DQ was prepared in the same manner by
treating [3H]DES with MnO2 in chloroform.
Irreversible binding assays
The cytosol ER was partially purified by a 50% ammonium sulfate
precipitation.14 The ammonium sulfate pellets were resuspended
with TEGM buffer, and 100 ␮L aliquots were incubated with 5 nM
[3H]E2, [3H]DES and [3H]DQ at 4°C for 2 h with or without a
200-fold excess of unlabeled DES (1 ␮M) in a total volume of 0.5
mL of TEGM buffer. Irreversible binding assays were performed
Tissue preparations and competitive binding assays
Competitive binding assays were carried out with minor modifi-
cation of a previously described procedure.13 Ovariectomized fe-
Figure 1 In vitro and in vivo metabolism of DES and DQ. DES is enzymatically oxidized to DQ by cytochrome P-450 or peroxidase and
chemically by activated MnO2. DQ is further rearranged to ZZ-DIEN.
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Diethylstilbestrol quinone: Chae et al.
Figure 2 HPLC Chromatography of DES and DQ. Chemical synthesis of DQ from DES was monitored by HPLC. The C-18 reversed
phase column was used to separate DES and DQ. The column was eluted with a mixture of acetonitrile and water. The solvent was
programmed from 50% acetonitrile in water to 100% acetonitrile in 30 min at a flow rate of 1 mL/min and the chromatogram was
monitored with a diode array UV detector.
Figure 3 Competitive binding analysis of DES, DQ and ZZ-DIEN with mouse uterine cytosol ER. Mouse uterine cytosol was incubated
with 5 nM of [3H]E2, and 0.1 to 100 nM of unlabelled ligands. After incubation for 18 h at 4°C the receptor was precipitated by addition
of 60% HAP. The precipitate was washed with buffer, and the radioactivity was counted. Numbers in parentheses are relative binding
affinities (RBA) which were calculated as describes in the Experimental. Shown are the mean values of two separate experiments
performed in duplicate.
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Figure 4 Covalent binding of DQ. Five nM 3H-DES and 3H-DQ were incubated with resuspended ammonium sulfate precipitated
mouse uterine cytosol with or without the 200-fold excess unlabeled DES in a total volume of 0.5 mL. After incubating for 18 h at 4°C,
the mixture was treated with DCC and 100 ␮L aliquots were placed on Whatman 3MM filter paper. A, C) Filter paper was counted
directly. B, D) Filter paper was treated with hot ethanol for 30 min and washed with ethanol/ether and counted. Total Bound: 3H-Labeled
ligands were incubated without unlabeled DES. NonSpecific Bound: 3H-Labeled ligands were incubated with 200-fold excess unlabeled
DES. Specific Bound: Calculated by subtracting non-specific bound from total bound.
152 Steroids, 1998, vol. 63, March

Diethylstilbestrol quinone: Chae et al.
Figure 5 Transfection assay of ER-dependent activation of estrogen-responsive CAT reporter gene. COS-1 cells were transfected with
both pcDNA3/wild type ER expression vector (25 ng/plate) and the ERET81CAT estrogen-responsive reporter vector (5 ␮g/plate), or the
ERET81CAT reporter vector alone, and treated with 1 nM of DES or DQ. All cells were cotransfected with ␤-galactosidase expression
vector (5 ␮g/plate) for normalization. Cells were treated with ethanol vehicle, DES (1 nM) or DQ (1 nM) in triplicate and the data
represent the mean Ϯ SE of two separate experiments. Numbers on top of the bar indicates the fold induction over the control vehicle.
according to the modification of boiling-ethanol disc assay.15
Unbound steroid was absorbed with dextran-coated charcoal
(DCC) for 5 min at 4°C, followed by centrifugation at 1000 ϫ g
for 10 min. Aliquots of the incubation mixture (100 ␮L) were
placed on a Whatman 3MM filter paper, dried by a stream of
nitrogen and the radioactivity was counted using Ecoscint O scin-
tillation cocktail. For the irreversible binding analysis, filter papers
were treated with boiling hot ethanol for 30 min, rinsed with an
ethanol/ether (1:1) mixture and the radioactivity was counted.
Counts from the unwashed filter papers were used to determine
specific binding deriving from non-covalent and covalently bound
ligand. Specific binding due to covalent ligand-ER bounds were
calculated from the washed filters.
with an estrogen responsive reporter construct (ERET81CAT)18
and a mouse ER expression vector (pcDNA3/wild type ER).
COS-1 cells were grown in Dulbecco’s Modified Eagle’s Medium
(DMEM without phenol red) supplemented with 10% charcoal
stripped fetal calf serum (sFCS) and 2 mM L-glutamine. Cells were
plated in 100-mm tissue culture plates at approximately 50%
confluence. Twenty-four h after plating, cells were transfected
with Caϩ2/PO4Ϫ2 precipitates containing pcDNA3/wild type ER
(25 ng/plate) and ERET81 CAT vector (5 ␮g/plate). All cells were
cotransfected with a ␤-galactosidase expression vector (pCH111, 5
␮g/plate) for normalization of transfection efficiencies. After in-
cubation at 37°C for 4 h, the cells were shocked for 30 s with
glycerol (15% in DMEM), washed with DMEM, and incubated
with vehicle (ethanol) or appropriate ligands in DMEM with 10%
sFCS. In some experiments, the cells were pre-treated for 15 min
with 10 nM of pure anti-estrogen ICI 182,780 before addition of
the ligands. After incubation with the ligands for 30 min at 37°C,
the media was aspirated and replaced with fresh DMEM media
containing 10% sFCS. Following incubation for 18 h at 37°C, the
Transfections and CAT assay
Transient transfections and chloramphenicol acetyltransferase
(CAT) assays were done as described previously.16 Using a Caϩ2
/
PO4Ϫ2 precipitation technique,17 COS-1 cells were transfected
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Figure 6 Effect of antiestrogen treatment on estrogen-responsive CAT activity in COS-1 cells. The COS-1 cells were transfected with
an ER expression vector, estrogen-responsive CAT and ␤-galactosidase vectors, and treated with 1 nM each of DES, DQ and ZZ-DIEN.
Prior to treatment with the ligands, the cells were pretreated for 15 min with 10 nM of pure anti-estrogen ICI 182,780. After incubation
for 30 min, incubation medium was removed and fresh DMEM with 10% sFCS medium without compounds was added. The ER
transactivation was assayed by CAT assay and normalized by ␤-galactosidase assay. Numbers on top of the bar indicates the fold
induction over the control vehicle. Data correspond to the mean Ϯ SE in triplicate and represent two separate experiments.
cells were harvested, pelleted, and lysed by freezing and thawing
the cells four times in 300 ␮L of 100 mM Tris (pH 7.8). Aliquots
of extracts (50 ␮L) were assayed for CAT and ␤-galactosidase
activity according to previously reported methods.19
These compounds produced typical competitive binding
curves although DQ (RBA ϭ 3.6) and ZZ-DIEN (RBA ϭ
0.3) exhibited significantly lower binding affinities than
DES (RBA ϭ 286).
To determine whether DQ binds to the ER irreversibly,
[3H]DQ was incubated with ammonium sulfate precipitated
mouse uterine cytosol for 18 h at 4°C with or without a
200-fold excess of unlabeled DES. For comparison, irre-
versible binding of [3H]E2 and [3H]DES was also deter-
mined. After treating the incubation mixture with DCC to
remove unbound ligands, aliquots were placed on Whatman
3MM filter paper and treated with hot ethanol for 30 min to
remove any reversibly bound ligands. Specifically bound
[3H]E2 and [3H]DES was effectively removed by the hot
ethanol treatment while approximately 30% of the specifi-
cally bound [3H]DQ was not washed out by the hot ethanol
treatment (Figure 4).
Results
DQ was synthesized for these studies by oxidation of DES
with activated manganese (IV) oxide (MnO2) in anhydrous
chloroform (Figure 1). HPLC analysis determined that the
formation of DQ occurred within 5 min at room tempera-
ture. Using this synthetic route and under these conditions,
DES (Rt ϭ 10.1 min) was completely converted to DQ (Rt
ϭ 5.2 min) and the two compounds were separable as
shown in the HPLC chromatogram (Figure 2). DQ in chlo-
roform was stable for at least 3 days at 4°C while kept in the
dark. No significant decomposition of DQ occurred for up
to a week when kept at Ϫ20°C in the dark.
In order to study the ability of DQ to activate ER depen-
dent genes, COS-1 cells were transiently transfected with an
Competitive binding experiments of DES, DQ and ZZ-
DIEN with mouse uterine cytosol ER are shown in Figure 3.
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Diethylstilbestrol quinone: Chae et al.
Figure 7 Dose curve of estrogenic activation of CAT gene by DES and DQ on CAT activity in COS-1 cells. The COS-1 cells were
transfected with an ER expression vector, estrogen-responsive CAT and ␤-galactosidase reporter vectors, and treated with increasing
concentrations of DES and DQ, and ethanol vehicle control. The CAT activity reported was normalized by ␤-galactosidase assay. Data
correspond to the mean Ϯ SE in triplicate and represent two separate experiments.
estrogen-responsive reporter construct (ERET81CAT) and
an ER expression vector (pcDNA3/wild type ER). In the
presence of ER, equimolar (1 nM) amounts of DES and DQ
induced similar increases (11-fold) in CAT activity whereas
ZZ-DIEN did not induce CAT activity (Figure 5). No trans-
activation was observed in the absence of cotransfected ER.
To further confirm that the transcriptional activation of
CAT by DES, DQ and ZZ-DIEN was mediated by ER,
transfected cells were also treated with 10-fold excess of a
pure antiestrogen, ICI 182,780.20 Pre-treating the cells with
ICI 182,780 blocked both DES- and DQ-induced CAT
activity (Figure 6). Figure 7 shows the concentration-
dependent increase in CAT activity after treating the ER
transfected cells with increasing concentrations of DES and
DQ.
DQ was converting to DES during incubation. Figure 8
displays the UV absorption spectrum of DQ at various times
immediately after addition to the cell culture media (COS-1
cells in sFCS/DMEM media) at 37°C. As DQ (␭max 312 and
345 nm) peaks decreased in this media, the ZZ-DIEN (␭
max
260 nm) peak increased. This rearrangement is the result of
a non-enzymatic process.21 The conversion of DQ to DES
(␭
250 and 282 nm) was not observed in this system.
max
Discussion
Oxidative metabolism of DES has been shown to follow a
variety of pathways.22 It has been postulated that the met-
abolic oxidation of DES to DQ and subsequent covalent
binding of DQ to protein and DNA might contribute to the
carcinogenicity and toxicity of DES.10 Therefore, we have
investigated ER binding of DES and DQ, and the capability
of these compounds to interact with ER and activate estro-
We were concerned that DQ might be converted back to
DES in the system. Therefore, a time course study of DQ in
the COS/FCS media was performed to determine whether
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Figure 8 The stability of DQ in sFCS DMEM media containing COS-1 cells. DQ (0.1 mM) was added to an incubation mixture of COS-1
cells in DMEM media with 10% sFCS at 37°C. Ultraviolet spectra scans were recorded at the indicated time intervals.
gen dependent gene activity in transiently transfected COS
cells.
gene was tested. COS cells were transfected with or without
the ER vector and an estrogen responsive gene. DQ (1 nM)
induced CAT activity of 10.1-fold over ethanol vehicle
control, compared to 11-fold for DES (1 nM). Thus, even
though DQ has a much lower binding affinity to ER, its
ability to induce transactivation of an ERE reporter gene is
similar to DES. The antiestrogen ICI 182,780 was able to
block these transactivations. Testing with and without the
ER and with a pure antiestrogen in the presence of the ER
proved that the observed estrogen response of the DQ is
through direct interactions with the ER. To determine
whether any activity of DQ was a result of the conversion
back to DES, the stability of DQ in the COS-1/10% sFCS/
DMEM media was monitored by UV scan. After 60 min of
incubation of DQ in this media containing COS-1 cells, as
shown in Figure 8, no conversion of DQ (␭max 312 and 345
However, since DES has a high binding affinity to ER
and is a potent inducer of estrogen dependent genes, it was
important to obtain a pure preparation of DQ without any
contamination by DES. Chemical synthesis of DQ from
DES was accomplished using a mild oxidizing agent, MnO2
in chloroform.12 This procedure caused complete oxidation
of DES to DQ without further oxidation to ZZ-DIEN. The
advantages of using MnO2 over the other oxidizing agents
are the purity of the product and the fact that the reaction
product can be obtained directly from the reaction mixture
without further separation or purification.
Once pure preparations of DQ were readily available, we
examined the ability of DES, DQ and ZZ-DIEN to interact
with ER. Previous studies showed that the mouse ER ex-
hibits stereospecific recognition of molecules,23 and that
both hydroxyl groups of stilbene estrogens are needed for
high ER binding.24 When DES was oxidized to DQ, the
competitive ER binding affinity decreased 80-fold as shown
in Figure 3. Since the ER has a much lower binding affinity
for ZZ-DIEN (RBA ϭ 0.3) than DES or DQ, any small
amounts of ZZ-DIEN in the DQ binding assays would have
exerted minimal effects on assessments of RBAs for DQ.
Initially, the low RBA of DQ for the ER suggested that
DQ might not be responsible for some of the deleterious
effects of DES. However, our subsequent studies demon-
strated that a significant percentage of specifically bound
DQ is covalently bound to ER. This covalent interaction
between DQ and ER could lead to persistent ER activation
and significant biological activity despite the low RBA of
DQ for the ER.
nm) to DES (␭
250 and 282 nm) was observed. Liehr
max
reported that the half-life of DQ is approximately 40 min in
water and 70 min in methanol.21 The present study con-
firmed previously reported findings. Although DQ rear-
ranged to ZZ-DIEN within 1 h of addition to the media, the
transactivation due to DQ was clearly not the result of the
formation of ZZ-DIEN as shown in Figure 5.
These findings indicate that despite a low RBA, DQ is
equally effective as DES at inducing gene activity. Our
data suggest this may be due to the ability of DQ to form
covalent bonds with ER. Covalent binding of DQ to ER
may contribute to the high gene activation and may be
related to the carcinogenicity of DES. One implication of
these studies is that weakly estrogenic compounds that
are reactive and capable of forming covalent bonds with
ER may have biological effects that are disproportionate
to their RBAs.
To determine if DQ binding to ER results in any induc-
tion of gene activity, the transactivation of an ERE reporter
156 Steroids, 1998, vol. 63, March

Diethylstilbestrol quinone: Chae et al.
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