Interaction of methoxychlor and related compounds with estrogen receptor α and β, and androgen receptor: Structure-activity studies

Article abstract of DOI:10.1124/mol.58.4.852

We previously demonstrated differential interactions of the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) with estrogen receptor α (ERα), ERβ, and the androgen receptor (AR). In this study, we characterize the ERα, ERβ, and

Full text of DOI:10.1124/mol.58.4.852

0026-895X/00/040852-07$3.00/0
MOLECULAR PHARMACOLOGY
Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 58:852–858, 2000
Vol. 58, No. 4
145/849160
Printed in U.S.A.
Interaction of Methoxychlor and Related Compounds with
Estrogen Receptor ␣ and ␤, and Androgen Receptor:
Structure-Activity Studies
KEVIN W. GAIDO, SUSAN C. MANESS, DONALD P. MCDONNELL, SHANGARA S. DEHAL, DAVID KUPFER, and
STEPHEN SAFE
Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina (K.W.G., S.C.M.); Department of Pharmacology and Cancer
Biology, Duke University Medical Center, Durham, North Carolina (D.P.M.); Department of Pharmacology and Molecular Toxicology, University
of Massachusetts Medical Center, Worcester, Massachusetts (S.S.D., D.K.); and Department of Veterinary Physiology and Pharmacology,
Texas A&M University, College Station, Texas (S.S.)
Received March 10, 2000; accepted June 15, 2000
This paper is available online at http://www.molpharm.org
ABSTRACT
We previously demonstrated differential interactions of the me- of 2,2-bis(p-hydroxyphenyl)-1,1-dichloroethylene, had ER␣ ag-
thoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichlo- onist activity and ER␤ and AR antagonist activity similar to
roethane (HPTE) with estrogen receptor ␣ (ER␣), ER␤, and the HPTE. The trihydroxy metabolite of methoxychlor displayed
androgen receptor (AR). In this study, we characterize the ER␣, only weak ER␣ agonist activity and did not alter ER␤ or AR
ER␤, and AR activity of structurally related methoxychlor me- activities. Replacement of the trichloroethane or dichloroethyl-
tabolites. Human hepatoma cells (HepG2) were transiently ene group with a methyl group resulted in a compound with
transfected with human ER␣, ER␤, and AR plus an appropriate ER␣ and ER␤ agonist activity that retained antiandrogenic ac-
steroid-responsive luciferase reporter vector. After transfection, tivities. This study identifies some of the structural requirements
cells were treated with various concentrations of HPTE or for ER␣ and ER␤ activity and demonstrates the complexity
structurally related compounds in the presence (for detecting involved in determining the mechanism of action of endocrine-
antagonism) and absence (for detecting agonism) of 17␤-es- active chemicals that simultaneously act as agonists or antag-
tradiol and dihydrotestosterone. The monohydroxy analog of onists through one or more hormone receptors.
methoxychlor, as well as monohydroxy and dihydroxy analogs
Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane]
Estrogenic responses are mediated through two separate
is a chlorinated hydrocarbon pesticide structurally similar to di- estrogen receptors, ER␣ and ER␤. These two receptors have
chlorodiphenyltrichloroethane [DDT; 1,1,1-trichloro-2,2-bis(chlo- homologous DNA and ligand binding regions (Kuiper and
rophenyl)ethane]. Like o,pЈ-DDT, methoxychlor is estrogenic in Gustafsson, 1997; Tremblay et al., 1997; Ogawa et al., 1998),
vivo (Bulger et al., 1978; Gray et al., 1989; Alm et al., 1996; Chapin and most compounds have similar binding affinities and
et al., 1997; Cummings, 1997; Hall et al., 1997). However, me-
thoxychlor has low affinity for the estrogen receptor (ER) and the
in vivo estrogenic activity is caused by metabolism to phenolic
estrogenic metabolites. The primary estrogenic metabolite of me-
thoxychlor is 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane
(HPTE). HPTE competes with estradiol for binding to ER, induces
ornithine decarboxylase and uterotrophic activity in ovariecto-
mized rats, and is approximately 100-fold more active than me-
thoxychlor (Bulger et al., 1978; Ousterhout et al., 1981; Shelby et
al., 1996).
transcriptional activities with ER␣ and ER␤ (Kuiper et al.,
1996, 1998; Mosselman et al., 1996; Tremblay et al., 1997).
We previously demonstrated that HPTE is an ER␣ agonist
and an ER␤ antagonist in HepG2 human hepatoma cells
transfected with estrogen-responsive reporter constructs
(Gaido et al., 1999). This unique activity of HPTE makes it an
ideal compound with which to evaluate the in vitro and in
vivo differences in ER-subtype dependent responses. We
have also shown that HPTE is an androgen receptor (AR)
antagonist in vitro (Maness et al., 1998). The differential
activity of HPTE with ER␣, ER␤, and AR may explain why
some of the responses induced by methoxychlor in vivo differ
from those induced by estradiol. For example, the ability of
The financial assistance of the National Institutes of Health (ES000834,
ES09106, and ES04917) and the Texas Agricultural Experiment Station is
gratefully acknowledged.
ABBREVIATIONS: DDT, dichlorodiphenyltrichloroethane; ER, estrogen receptor; AR, androgen receptor; TLC, thin-layer chromatography; E2,
17␤-estradiol; GC-MS, gas chromatography-mass spectrometry; p,pЈ-DDE, 2,2-bis(p-hydroxyphenyl)-1,1-dichloroethylene; C3, complement 3;
Luc, luciferase; CMV, cytomegalovirus; CPRG, chlorophenol red-␤-D-galactopyranoside; HPTE, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane;
DHT, dihydrotestosterone; THC, tetrahydrochrysene.
852

Structure-Activity Studies with Steroid Hormone Receptors
853
methoxychlor to act as an ER antagonist in the ovary (Hall et activity, cells were transfected with a human ER␤ expression plas-
mid, a C3-Luc reporter plasmid, and CMV-␤-galactosidase reporter
plasmid (Gaido et al., 1999; Hall and McDonnell, 1999). For detection
of AR activity, cells were transfected with a human AR expression
plasmid, plus an androgen-responsive MMTV-Luc reporter plasmid,
and CMV-␤-galactosidase reporter plasmid (Maness et al., 1998).
Transfected cells were rinsed with PBS and dosed with various
concentrations of test chemical and dimethyl sulfoxide (vehicle con-
trol; Sigma) in complete medium. After a 24 h incubation, cells were
rinsed with PBS and lysed with 65 ␮l of lysing buffer (25 mM
Tris-phosphate, pH 7.8, 2 mM 1,2-diaminocyclohexane-N,N,NЈ,NЈ-
al., 1997) may be caused by the high level of ER␤ expression
relative to ER␣ in this tissue (Saunders et al., 1997).
The physiological consequences of a chemical that is an
ER␣ agonist, an ER␤ antagonist, and an AR antagonist are
unknown, and HPTE can serve as a model for investigating
the effects of an agent that modulates multiple endocrine
pathways. Additional studies with HPTE and structural an-
alogs may lead to further insights on ligand specificity for
ER␣, ER␤, and AR. Therefore we compared the ER␣, ER␤,
and AR activity of HPTE and structural analogs and show tetraacetic acid, 10% glycerol, 0.5% Triton X-100, 2 mM dithiothre-
itol). Lysate was divided into two 96-well plates for luciferase and
␤-galactosidase determination.
that some chemicals similar in structure to HPTE also dem-
onstrate unique ER␣, ER␤, and AR activity.
Luciferase activity was determined by adding 100 ␮l of Luc assay
reagent (Promega, Madison, WI) to the first 96-well plate containing
20 ␮l of lysate. Luminescence was determined immediately using a
ML3000 microtiter plate luminometer (Dynatech Laboratories,
Chantilly, VA).
Materials and Methods
Chemicals. HPTE was synthesized by dissolving 1 g of methoxy-
chlor (Aldrich Chemical Co., Milwaukee, WI) in 100 ml of methylene
chloride and then treating with excess boron tribromide in methyl-
ene chloride (Aldrich) for 24 h. Water (5 ml) was carefully added, and
crude HPTE was isolated in methylene chloride. The residue (0.8 g)
was purified by preparative thin-layer chromatography (TLC). The
resulting HPTE was Ͼ97% pure as determined by gas-liquid chro-
matography.
Monohydroxymethoxychlor was synthesized by dissolving 1.0 g of
methoxychlor in methylene chloride. Approximately 1.5 mol equiva-
lents of boron dibromide in methylene chloride was slowly added
over a period of 1 to 2 h. The progress of demethylation was moni-
tored by TLC. The monohydroxymethoxychlor metabolite was iso-
lated by preparative TLC using hexane/acetone (92:8) as solvent.
Yields of 250 to 300 mg were obtained and the products were greater
than 98% pure as determined by gas chromatography-mass spec-
trometry (GC-MS).
Trihydroxymethoxychlor and the corresponding trimethoxyme-
thoxychlor were synthesized by ChemSyn Labs (Lenexa, KS). Dime-
thoxy-DDE was synthesized by dissolving 1.0 g of methoxychlor in
dimethyl sulfoxide. Anhydrous sodium bicarbonate (3.0 g) was added
and the mixture was heated at 140°C for 1 h. The mixture was
diluted with water and the dimethoxy-DDE product was isolated by
extraction with chloroform. The crystalline residue from the chloro-
form extract (0.75 g) was greater than 98% pure as determined by
GC-MS. Dihydroxy-DDE was prepared from 2,2-bis(p-hydroxyphen-
yl)-1,1-dichloroethylene (p,pЈ-DDE) following the same procedure as
described above for HPTE. Dihydroxy-DDE was greater than 98%
pure as determined by GC-MS. Monohydroxy-DDE was prepared
from p,pЈ-DDE following the same procedure as described above for
monohydroxymethoxychlor. Monohydroxy-DDE was greater than
98% pure as determined by GC-MS. All other chemicals were ob-
tained from Sigma Chemical Co. (St. Louis, MO) and were Ն97%
pure.
␤-Galactosidase activity was determined by adding 20 ␮l of ␤-ga-
lactosidase assay reagent to 30 ␮l of lysate in the second 96-well
plate. ␤-Galactosidase assay reagent consisted of a 4 mg/ml solution
of chlorophenol red-␤-D-galactopyranoside (CPRG; Sigma) in 150 ␮l
of CPRG buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1
mM MgSO4, 50 mM ␤-mercaptoethanol, pH 7.8) Absorbance at 570
nm was determined over a 30 min period using a Vmax kinetic
microplate reader (Molecular Devices, Menlo Park, CA).
HepG2 cells lack detectable levels of endogenous ER␣, ER␤, and
AR and in the absence of transfected receptor, Luc activity remains
below the level of detection (data not shown). Background activity
after receptor transfection averaged 150 Ϯ 56 normalized Luc units
with ER␣, 31 Ϯ 6 normalized Luc units with ER␤, and 5 Ϯ 1
normalized Luc units with AR. We have previously confirmed by
Western analysis that ER␣ and ER␤ are expressed at equal concen-
trations under the conditions of our assay (Hall and McDonnell,
1999).
Statistical Analysis. Unless otherwise noted, values presented
in this study represent the means Ϯ S.E. resulting from at least
three separate experiments with triplicate wells for each treatment
dose. Dose-response data were analyzed using the sigmoidal dose-
response function of the graphical and statistical program Prism
(GraphPad, San Diego, CA).
Results
We compared the activity of HPTE and structural analogs
in HepG2 cells transfected with expression vectors for human
ER␣, ER␤, and AR along with the appropriate reporter plas-
mid (Fig. 1). HepG2 cells were dosed with set concentrations
of chemical alone and in combination with an inducing dose
of either 17␤-estradiol (E2) or dihydrotestosterone (DHT) for
determining antagonistic activity with ER␣/␤ and AR, re-
spectively. HPTE (Fig. 1C) exhibited ER␣ agonist, and ER␤
and AR antagonist activity as described previously (Maness
et al., 1998; Gaido et al., 1999). HPTE does display some
Plating and Transfection. Transfection experiments were per-
formed as described previously (Maness et al., 1998; Gaido et al.,
1999). HepG2 human hepatoma cells (ATCC, Rockville, MD) were
plated in triplicate in 24-well plates (Falcon Plastics, Oxnard, CA) at
a density of 105 cells/well in complete medium consisting of phenol partial ER␤ agonist activity of approximately 13% of that
red-free Eagle’s minimal essential medium (GIBCO/BRL, Grand Is-
land, NY) supplemented with 10% resin-stripped fetal bovine serum
(Hyclone, Logan, UT), 2% L-glutamine, and 0.1% sodium pyruvate.
Cells were incubated overnight at 37°C in a humidified atmosphere
of 5% CO2/air and then transfected after the Superfect procedure
(Qiagen, Valencia, CA) with three plasmids. For detection of ER␣
activity, cells were transfected with human ER␣ expression plasmid,
plus an estrogen-responsive complement 3-luciferase (C3-Luc) re-
porter plasmid, and a constitutively active cytomegalovirus (CMV)-
␤-galactosidase reporter plasmid (transfection and toxicity control)
obtained with a maximally inducing dose of estradiol (Man-
ess et al., 1998). The monohydroxy metabolite of methoxy-
chlor, as well as the mono- and dihydroxy analogs of p,pЈ-
DDE (Fig. 1, B, H, and I), also had ER␣ agonist and ER␤ and
AR antagonist activity. Bisphenol A exhibited ER␣ and ER␤
agonist activity but did not have antiandrogenic activity (Fig.
1K). Replacement of the trichloromethyl of HPTE or dichlo-
romethylene group of dihydroxy-DDE results in a conversion
from ER␤ antagonist activity to full ER␤ agonist activity but
(Tzukerman et al., 1994; Gaido et al., 1999). For detection of ER␤ retains ER␣ agonist and AR antagonist activity (Fig. 1, C and

854
Gaido et al.
Fig. 1. ER␣, ER␤, and AR activity of methoxychlor analogs. HepG2 cells were transiently transfected with expression plasmids for human ER␣, ER␤, and
AR plus C3-Luc and a constitutively active ␤-galactosidase expression plasmid (transfection and toxicity control). Cells were treated with increasing
concentrations of methoxychlor analogs alone for detecting agonist activity (hatched bars) and with an inducing dose of either estradiol or DHT for detecting
antagonist activity (shaded bars). Luciferase activity was normalized to ␤-galactosidase activity. Values represent the means Ϯ S.E. of three separate
experiments and are presented as percentage response, with 100% activity defined as the activity achieved with 10Ϫ7 M estradiol for ER␣ and ER␤, and 10Ϫ7
M DHT for AR. The abscissa represents log molar concentration. Agonists cause an increase in percentage response (rising hatched bars) with increasing
concentration, whereas antagonists cause a decrease in percentage response (declining shaded bars) with increasing concentration.

Structure-Activity Studies with Steroid Hormone Receptors
855
I versus L). Trimethoxy and trihydroxy ring substituted com- cies. Monohydroxy-DDE, bishydroxyphenylmethane, and
pounds (Fig. 1, D and E) exhibited minimal ER␣ agonist bishydroxyphenylethane were approximately 3- to 5-fold less
activity and did not affect ER␤ or AR-dependent responses.
Concentration-response curves for selected ER␣ and ER␤
agonists are presented in Fig. 2, A and B. EC50 values for
ER␣ and ER␤ agonist activity are presented in Table 1.
HPTE and dihydroxy-DDE were most potent as ER␣ agonists
and were approximately 17- and 25-fold less potent, respec-
tively, than estradiol. HPTE and dihydroxy-DDE were fol-
lowed in ER␣ agonist potency by monohydroxy methoxychlor,
bisphenol A, monohydroxy-DDE, bishydroxyphenylmethane,
and bishydroxyphenylethane. Bishydroxyphenylmethane
and bishydroxyphenylethane were equally potent as ER␤
agonists and were approximately 285-fold less potent than
estradiol.
We characterized the ER␤ antagonist activity of selected
compounds in HepG2 cells by determining the effect of vari-
ous concentrations across a complete estradiol dose-response
range (Fig. 3, A–C). Each of the tested compounds caused
parallel shifts in the estradiol dose-response curve, indicat-
ing competitive antagonism. Schild regression analyses were
performed and equilibrium dissociation (KB) values deter-
mined (Table 1). HPTE, monohydroxy methoxychlor, mono-
hydroxy-DDE, and dihydroxy-DDE demonstrated relatively
similar antagonist potencies.
potent than HPTE, dihydroxy-DDE, and p,pЈ-DDE.
Discussion
ER␣ and ER␤ bind structurally diverse classes of chemi-
cals, and it is difficult to compare structure-dependent recep-
tor binding or transactivation between chemical classes. In
contrast, structure-activity correlations within a single struc-
tural class such as the triphenylethanes can be used to de-
sign ER agonists and antagonists (e.g., tamoxifen) for clinical
applications as selective estrogen receptor modulators. For a
series of mono-, di-, and trihydroxyphenyl ethane/ethylene
analogs structurally related to methoxychlor, the order of
potency as ER␣ agonists was dihydroxyphenyl Ͼ monohy-
droxyphenyl Ͼ trihydroxyphenyl, suggesting that the opti-
mal structure for ER␣ agonist activity contained two p-hy-
droxyphenyl groups substituted at
a single ethane or
ethylene carbon atom (i.e., bis-substitution). In contrast, the
fully methylated metabolites (e.g., Fig. 1, A versus C; Fig. 1,
G versus D) were significantly less active as ER␣ agonists;
this is consistent with previous studies with methoxychlor
and HPTE (Bulger et al., 1978; Ousterhout et al., 1981;
Shelby et al., 1996). A similar pattern was observed for the
substituted diphenylmethane analogs even though only a
limited number of these compounds were tested (Fig. 1, J and
N versus M).
Similar experiments were performed to characterize AR
antagonist activity (Fig. 4, Table 1). HPTE, dihydroxy-DDE,
and p,pЈ-DDE demonstrated similar AR antagonist poten-
With few exceptions (Fig. 1, K and L), the bishydroxy/
methoxyphenyl ethane or ethylene analogs were not signifi-
cant ER␤ agonists, and only two bishydroxyphenylmethanes
(Fig. 1, J and N) induced measurable ER␤-dependent re-
porter gene activity. Thus, our results demonstrate that this
series of bishydroxyphenyl-substituted ethanes, ethylenes,
and methanes are preferential ER␣ agonists and exhibit
weak to nondetectable ER␤ agonist activity. These results
are unique because previous studies for ER subtype-depen-
dent ligand binding and transactivation report similar ER␣
and ER␤ activity for various structural classes of estrogenic
compounds (Kuiper et al., 1996, 1998; Mosselman et al.,
1996; Tremblay et al., 1997).
The results of our studies also demonstrate that methoxy-
chlor and structurally related analogs exhibit minimal ER␣
antagonist activity but that three compounds (Fig. 1, B, H,
TABLE 1
A comparison of the agonist and antagonist potencies of methoxychlor
analogs with ER␣, ER␤, and AR
ER␣
EC50
ER␤
AR
Chemicals
KB
EC50
ϫ 10Ϫ8
KB
M
EC50
1.0
KB
Estradiol
DHT
0.3
0.7
HPTE
5.1a
19.8
67.0
7.4
3.0a
7.2
8.5
5.1
31.6
Monohydroxy-methoxychlor
Monohydroxy-DDE
Dihydroxy-DDE
100.0
29.4
142.0
136.0
36.9b
Bishydroxyphenylmethane 150.0
200.0
210.0
Bishydroxyphenylethane
p,pЈ-DDE
Bisphenol A
160.0
Fig. 2. Dose response of selected methoxychlor analogs with ER␣ (A) and
ER␤ (B). Experiments were performed as described in Fig. 1. Luciferase
activity was normalized to ␤-galactosidase activity. Values represent the
means Ϯ S.E. of three separate experiments. f, E2; F, monohydroxy-
methoxychlor; ‚, monohydroxy-DDE; {, dihydroxy-DDE; ƒ, bishydroxy-
phenyl methane; Œ, bishydroxyphenyl ethane.
64.0c
89.0c
a From Gaido et al. (1999).
b From Maness et al. (1998).
c From Gould et al. (1998).

856
Gaido et al.
and I) in addition to HPTE (Fig. 1C) are highly effective ER␤ antiandrogens and exhibited activity similar to that observed
antagonists. Structural features required for this response
include bis(4-hydroxyphenyl) or bis(4-hydroxyphenyl),(4-me-
thoxyphenyl) groups attached to chlorine-substituted ethane/
ethylene moieties. Additional compounds are required to
more accurately define structural requirements for ER␤ an-
tagonist activities; however, our results clearly demonstrate
remarkable structure-dependent differences among these
compounds for activity as ER␤ antagonists.
for p,pЈ-DDE. Interestingly, both p,pЈ-DDE (Fig. 1F) and
dihydroxy-DDE (Fig. 1I) exhibited similar antiandrogenic
activities, and the interchange of two p-chloro and two p-
hydroxyl substituents had minimal effects on this AR re-
sponse. In contrast, the two p-hydroxyl groups (but not p-
chloro substituents) conferred both ER␣ agonist and ER␤
antagonist activity on dihydroxy-DDE, demonstrating that
subtle substituent changes can affect some but not all ligand-
activated hormone receptor action.
Several compounds investigated in this study exhibited
antiandrogenic activity. Four analogs that were ER␤ antag-
onists (Fig. 1, B, C, H, and I) were among the most active
Methoxy-DDE and monohydroxy-DDE are impurities in
technical grade methoxychlor (Bulger et al., 1985), and dihy-
droxy-DDE is formed during the metabolism of methoxychlor
in mice (Kapoor et al., 1970). HPTE, monohydroxy-methoxy-
chlor, monohydroxy-DDE, and dihydroxy-DDE have previ-
ously been shown to compete with estradiol for ER binding in
vitro and demonstrate uterotropic activity in vivo (Bulger et
al., 1978, 1985; Ousterhout et al., 1981). Thus, exposure to
methoxychlor results in a complex interaction of multiple
metabolites with different activities at ER␣, ER␤, and AR.
The molecular mechanism by which a ligand can act as an
ER␣ agonist and an ER␤ antagonist is of both toxicological
and pharmacological interest. The overall structure of the
ER␤ ligand-binding domain is very similar to that of ER␣
(Pike et al., 1999), and most compounds demonstrate similar
binding affinities and transcriptional activities with ER␣ and
ER␤ (Kuiper et al., 1996, 1997; Mosselman et al., 1996;
Tremblay et al., 1997). The helix 12 region present on both
receptors plays an important role in the mechanism of ER
action (Darimont et al., 1998). This region folds over the
ligand binding pocket and exposes a region on both receptors
involved in coactivator binding. ER␣ and ER␤ antagonists
such as raloxifene and hydroxytamoxifen contain bulky con-
stituents that reposition helix 12 and block receptor interac-
tion with coactivators (Brzozowski et al., 1997; Pike et al.,
1999). HPTE analogs used in this study do not have substitu-
ents of the size and character of raloxifene and consequently
less likely to physically reposition helix 12. However, X-ray
crystallography and sequence analysis comparison of the li-
gand-binding domains of ER␣ and ER␤ suggest that the
agonist orientation of helix 12 in ER␤ may be unstable and
thus easier to antagonize than ER␣ (Pike et al., 1999). The
ER␣ agonist/ER␤ antagonists identified in this study may be
able to stabilize helix 12 in the agonist orientation for ER␣
but not for ER␤. X-ray crystallographic studies are needed to
confirm this hypothesis.
The R,R-enantiomer of tetrahydrochrysene (R,R-THC) has
also recently been shown to have differential ER␣ and ER␤
activity (Meyers et al., 1999; Sun et al., 1999). Like HPTE,
R,R-THC behaves as an ER␣ agonist and an ER␤ antagonist.
In contrast, the S,S-enantiomer (S,S-THC) is an agonist with
both ER␣ and ER␤. The equilibrium dissociation value (KB)
for R,R-THC has not been determined, and whether R,R-
THC is an ER␤ competitive antagonist remains to be dem-
onstrated. THC compounds differ considerably in structure
from the methoxychlor analogs presented in this study, and
this class of compounds will provide additional information
regarding the ligand specificity of ER␣ and ER␤ binding and
transactivation.
Fig. 3. Effect of various concentrations of methoxychlor analogs on an
estradiol dose-response curve with ER␤. Experiments were performed as
described in Fig. 1 with 10Ϫ10 to 10Ϫ5 M E2 either alone or in combination
with the indicated concentrations of monohydroxy-methoxychlor (A), mo-
nohydroxy-DDE (B), and dihydroxy-DDE (C). Luciferase activity was
normalized to ␤-galactosidase activity. Values represent the means of
three separate experiments.
Less is known about the mechanism of AR antagonism by
AR ligands. AR antagonists are generally thought to prevent

Structure-Activity Studies with Steroid Hormone Receptors
857
Fig. 4. Effect of various concentrations of methoxychlor analogs on a DHT dose-response curve with AR. Experiments were performed as described
in Fig. 1 with 10Ϫ9 to 10Ϫ5 M DHT either alone or in combination with the indicated concentrations of HPTE (A), bishydroxyphenylmethane (B),
bishydroxyphenylethane (C), monohydroxy-DDE (D), and dihydroxy-DDE (E). Luciferase activity was normalized to ␤-galactosidase activity. Values
represent the means of three separate experiments.
Acknowledgments
or reduce binding of AR to DNA (Kelce et al., 1995, 1998).
However, the specific mechanisms responsible for this inhi-
bition of AR-DNA binding remain unknown.
We thank Dr. Paul Foster, Dr. Chris Corton, and Dr. Katrina
Waters for critical review of the manuscript and Dr. Barbara Kuyper
for editorial assistance.
Much still remains to be determined about the precise roles
of ER␣, ER␤, and AR in reproductive development and en-
docrine function, especially in humans; the physiological con-
sequences of exposure to chemicals that are ER␣ agonists,
ER␤ antagonists, and AR antagonists are unknown. HPTE
and its structural analogs give us further insights into the
ligand specificity of ER␣, ER␤, and AR and serve as model
chemicals for investigating ER␣, ER␤, and AR steroid hor-
mone receptor interactions.
References
Alm H, Tiemann U and Torner H (1996) Influence of organochlorine pesticides on
development of mouse embryos in vitro. Reprod Toxicol 10:321–326.
Brzozowski AM, Pike A, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L,
Greene GL, Gustafsson JA and Carlquist M (1997) Molecular basis of agonism and
antagonism in the oestrogen receptor. Nature (Lond) 389:753–758.
Bulger WH, Feil VJ and Kupfer D (1985) Role of hepatic monooxygenases in gener-
ating estrogenic metabolites from methoxychlor and from its identified contami-
nants. Mol Pharmacol 27:115–124.
Bulger WH, Muccitelli RM and Kupfer D (1978) Studies on the in vivo and in vitro

858
Gaido et al.
estrogenic activities of methoxychlor and its metabolites. Role of hepatic mono-
oxygenase in methoxychlor activation. Biochem Pharmacol 27:2417–2423.
Chapin RE, Harris MW, Davis BJ, Ward SM, Wilson RE, Mauney MA, Lockhart AC,
Smialowicz RJ, Moser VC, Burka LT, Collins BJ, Haskins EA, Allen JD, Judd L,
Purdie WA, Harris HL, Lee CA and Corniffe GM (1997) The effects of perinatal/
juvenile methoxychlor exposure on adult rat nervous, immune, and reproductive
system function. Fundam Appl Toxicol 40:138–157.
Cummings AM (1997) Methoxychlor as a model for environmental estrogens. Crit
Rev Toxicol 27:367–379.
Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD,
Fletterick RJ and Yamamoto KR (1998) Structure and specificity of nuclear re-
ceptor-coactivator interactions. Genes Dev 12:3343–3356.
Gaido KW, Leonard LS, Maness SC, Hall JM, McDonnell DP, Saville B and Safe S
(1999) Differential interaction of the methoxychlor metabolite 2,2-bis-(p-
hydroxyphenyl)-1,1,1-trichloroethane with estrogen receptors alpha and beta. En-
docrinology 140:5746–5753.
Gould JC, Leonard LS, Maness SC, Wagner BL, Conner K, Zacharewski T, Safe S,
McDonnell DP and Gaido KW (1998) Bisphenol A interacts with the estrogen
receptor alpha in a distinct manner from estradiol. Mol Cell Endocrinol 142:203–
214.
Gray LE Jr, Ostby J, Ferrell J, Rehnberg G, Linder R, Cooper R, Goldman J, Slott V
and Laskey J (1989) A dose-response analysis of methoxychlor-induced alterations
of reproductive development and function in the rat. Fundam Appl Toxicol 12:92–
108.
Hall JM and McDonnell DP (1999) The estrogen receptor beta-isoform (ERbeta) of
the human estrogen receptor modulates ER␣ transcriptional activity and is a key
regulator of the cellular response to estrogens and antiestrogens. Endocrinology
140:5566–5578.
Kuiper G and Gustafsson JA (1997) The novel estrogen receptor-beta subtype:
Potential role in the cell- and promoter-specific actions of estrogens and anti-
estrogens. FEBS Lett 410:87–90.
Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der
Burg B and Gustafsson JA (1998) Interaction of estrogenic chemicals and phy-
toestrogens with estrogen receptor beta. Endocrinology 139:4252–4263.
Maness SC, McDonnell DP and Gaido KW (1998) Inhibition of androgen receptor-
dependent transcriptional activity by DDT isomers and methoxychlor in HepG2
human hepatoma cells. Toxicol Appl Pharmacol 151:135–142.
Meyers MJ, Sun J, Carlson KE, Katzenellenbogen BS and Katzenellenbogen JA
(1999) Estrogen receptor subtype-selective ligands: asymmetric synthesis and
biological evaluation of cis- and trans-5,11-dialkyl-5,6,11,12-tetrahydrochrysenes.
J Med Chem 42:2456–2468.
Mosselman S, Polman J and Dijkema R (1996) ER beta: identification and charac-
terization of a novel human estrogen receptor. FEBS Lett 392:49–53.
Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y and Muramatsu
M (1998) The complete primary structure of human estrogen receptor beta (hER
beta) and its heterodimerization with ER alpha in vivo and in vitro. Biochem
Biophys Res Commun 243:122–126.
Ousterhout J, Struck RF and Nelson JA (1981) Estrogenic activities of methoxychlor
metabolites. Biochem Pharmacol 30:2869–2871.
Pike ACW, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom O, Ljung-
gren J, Gustafsson JK and Carlquist M (1999) Structure of the ligand-binding
domain of oestrogen receptor beta in the presence of a partial agonist and a full
antagonist. EMBO J 18:4608–4618.
Saunders P, Maguire SM, Gaughan J and Millar MR (1997) Expression of oestrogen
receptor beta (ER beta) in multiple rat tissues visualised by immunohistochemis-
try. Endocrinology 154:R13–16.
Shelby MD, Newbold RR, Tully DB, Chae K and Davis VL (1996) Assessing envi-
ronmental chemicals for estrogenicity using a combination of in vitro and in vivo
assays. Environ Health Perspect 104:1296–1300.
Hall DL, Payne LA, Putnam JM and Huet-Hudson YM (1997) Effect of methoxychlor
on implantation and embryo development in the mouse. Reprod Toxicol 11:703–
708.
Kapoor IP, Metcalf RL, Nystrom RF and Sangha GK (1970) Comparative metabolism
of methoxychlor, methiochlor, and DDT in mouse, insects, and in a model ecosys-
tem. J Agric Food Chem 18:1145–1152.
Kelce WR, Gray LE and Wilson EM (1998) Antiandrogens as environmental endo-
crine disruptors. Reprod Fertil Dev 10:105–111.
Kelce WR, Stone CR, Laws SC, Gray LE, Kemppainen JA and Wilson EM (1995)
Persistent DDT metabolite p,pЈ-DDE is a potent androgen receptor antagonist.
Nature (Lond) 375:581–585.
Sun J, Meyers M, Fink B, Rajendran R, Katzenellenbogen J and Katzenellenbogen
B (1999) Novel ligands that function as selective estrogens or antiestrogens for
estrogen receptor␣ or estrogen receptor␤. Endocrinology 140:800–804.
Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F and
Giguere V (1997) Cloning, chromosomal localization, and functional analysis of the
murine estrogen receptor beta. Mol Endocrinol 11:353–365.
Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike
JW and McDonnell DP (1994) Human estrogen receptor transactivational capacity
is determined by both cellular and promoter context and mediated by two func-
tionally distinct intramolecular regions. Mol Endocrinol 8:21–30.
Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S and
Gustafsson JA (1997) Comparison of the ligand binding specificity and transcript
tissue distribution of estrogen receptors alpha and beta. Endocrinology 138:863–
870.
Kuiper G, Enmark E, Peltohuikko M, Nilsson S and Gustafsson JA (1996) Cloning of
a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci
USA 93:5925–5930.
Send reprint requests to: Dr. Kevin W. Gaido, CIIT, P.O. Box 12137,
Research Triangle Park, NC 27709. E-mail: gaido@ciit.org