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and ß1
Chemical Industry Institute of Toxicology (K.W.G., L.S.L., S.C.M.), Research Triangle Park, North Carolina 27709; the Department of Pharmacology and Cancer Biology, Duke University Medical Center (J.M.H., D.P.M.), Durham, North Carolina 27710; and the Department of Veterinary Physiology and Pharmacology, Texas A&M University (B.S., S.S.), College Station, Texas 77843
Address all correspondence and requests for reprints to: Dr. Kevin W. Gaido, Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, North Carolina 27709. E-mail: gaido{at}ciit.org
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and estrogen receptor ß (ERß).
Human hepatoma cells (HepG2) were transiently transfected with either
human or rat ER or ERß plus an estrogen-responsive, complement
3-luciferase construct containing a complement 3 gene promoter sequence
linked to a luciferase reporter gene. After transfection, cells were
treated with various concentrations of HPTE in the presence (for
detecting antagonism) or absence (for detecting agonism) of
17ß-estradiol. HPTE was a potent ER agonist in HepG2 cells, with
EC50 values of approximately 5 x 10-8
and 10-8 M for human and rat ER,
respectively. In contrast, HPTE had minimal agonist activity with
either human or rat ERß and almost completely abolished
17ß-estradiol-induced ERß-mediated activity. Moreover, HPTE behaved
as an ER agonist and an ERß antagonist with other
estrogen-responsive promoters (ERE-MMTV and vtERE) in HepG2 and HeLa
cells. This study demonstrates the complexity involved in determining
the mechanism of action of endocrine-active chemicals that may act as
agonists or antagonists through one or more hormone receptors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogenic activity is mediated by ligand binding to specific
intracellular proteins known as ERs (12). Ligand binding induces
conformational changes in the receptor, enabling the bound receptor
complex to interact with specific sites on DNA. Once bound to DNA the
ligand receptor complex alters expression of estrogen-responsive genes,
resulting in tissue-specific estrogenic responses. Until recently it
was thought that all estrogenic response occurred through a single
receptor, now termed ER. However, the identification of a second ER,
ERß (13), has prompted intensive research on the overlapping and
differential roles of these two receptors in mediating estrogenic
responses.
ER and ERß share a number of common physical and functional
properties. The DNA- and ligand-binding domains are highly homologous
between the two receptors (13, 14, 15). ER and ERß also demonstrate
many similarities in ligand binding affinities and regulation of gene
expression (14, 16, 17, 18, 19). Trans-activation studies using
17ß-estradiol (E2)-responsive constructs
transfected into mammalian cell lines have shown that ERß, like
ER, mediates the effects of E2 in a
dose-dependent manner, and ERß trans-activation is induced
by most ER agonists and blocked by ER antagonists (14, 16, 17, 19).
There are some reported differences in the transcriptional activities
of these two ER subtypes. Agonist activity of tamoxifen is selectively
observed with ER, but not ERß, in transiently transfected MCF-7
cells (20). In addition, E2 activates
ER-dependent transcription and inhibits ERß-dependent
transcription at activating protein-1 sites in transfected HeLa,
Ishikawa, and MCF-7 cells (21). Together, these studies demonstrate
some distinct differences in trans-activational mechanisms
between ER and ERß. Thus, it is important to characterize
interactions of hormonally active environmental chemicals with both
ER and ERß when trying to determine their potential to modulate
endocrine function.
Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenol)-ethane] has been of interest in our laboratories because of its well characterized estrogenic effects both in vitro and in vivo. Methoxychlor is a chlorinated hydrocarbon pesticide structurally similar to DDT that was introduced commercially for insect control in the 1950s and is still in use. Although it is structurally related to DDT, methoxychlor has an advantage in that it is more readily metabolized and excreted by mammalian systems and does not accumulate or bioconcentrate in fatty tissue (22). Like DDT, methoxychlor is estrogenic in vivo. Methoxychlor is uterotropic in the ovariectomized rat and can cause adverse developmental and reproductive effects in laboratory animals (23, 24, 25, 26, 27, 28).
Methoxychlor is metabolized in the liver by O-demethylation
to polar mono- and bis-phenolic metabolites (23). The bisphenolic
metabolite of methoxychlor
2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) is
approximately 100-fold more active at ER than methoxychlor. HPTE
competes with E2 for binding to ER and induces
ornithine decarboxylase and uterotropic activity in ovariectomized rats
(23, 29, 30). Conversion of methoxychlor to HPTE is generally
considered to be a pathway for metabolic activation into a more potent
estrogen. Although the interaction of HPTE with ER has been
characterized (30), no data on the interaction of this compound with
ERß have been reported.
In our studies we compared the activities of HPTE at ER and ERß
and show that HPTE is primarily an ER-specific agonist and an ERß
antagonist. Our results may lead not only to a better understanding of
the mechanism of methoxychlor toxicity but also to the identification
of additional ER- and ERß-specific agents.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plating and transfection
HepG2 human hepatoma cells (American Type Culture Collection, Manassas, VA) 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 red-free Eagle’s
MEM (Life Technologies, Inc./BRL, Grand Island, NY)
supplemented with 10% resin-stripped FBS (HyClone Laboratories, Inc., 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 following
the SuperFect procedure (QIAGEN, Valencia, CA) with three
plasmids (31): 1) various concentrations of receptor plasmid encoding
either human or rat ER (32, 33) or ERß (16); 2) 405 ng/well
complement 3-luciferase (C3-Luc), mouse mammary tumor virus (MMTV)-luc,
or estrogen response element-thymidine kinase-luciferase (ERE-TK-luc)
reporter plasmid (32, 34); and 3) 10 ng/well pCMVß-gal plasmid
(transfection control and for monitoring for toxicity; CMV,
cytomegalovirus; ß-gal, ß-galactosidase) 32). Transfected cells
were then rinsed with PBS and treated with various concentrations of
test chemical or dimethylsulfoxide (vehicle control; Sigma Chemical Co.) in complete medium. After a 24-h incubation,
treated cells were rinsed with PBS and lysed with 65 µl lysing buffer
[25 mM Tris-phosphate (pH 7.8), 2 mM
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic
acid, 10% glycerol, 0.5% Triton X-100, and 2 mM
dithiothreitol]. Lysate was divided into 2 96-well plates for
luciferase and ß-galactosidase determinations.
Luciferase assay
Luciferase assay reagent (100 µl; Promega Corp.,
Madison, WI) was added to 20 µl lysate, and luminescence was
determined immediately using an ML3000 microtiter plate luminometer
(Dynatech Corp., Chantilly, VA).
ß-Galactosidase assay
Twenty microliters of a 4 mg/ml solution of chlorophenol
red-ß-D-galactopyranoside (Sigma Chemical Co.) and 150 µl chlorophenol
red-ß-D-galactopyranoside buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1
mM MgSO4, and 50 mM
ß-mercaptoethanol, pH 7.8) were added to 30 µl lysate. Absorbance
at 570 nm was determined over a 30-min period using a Vmax
kinetic microplate reader (Molecular Devices, Menlo Park,
CA).
Competitive binding assay
The assay was performed as previously described (33). Serial
dilutions of E2 were prepared in 10
mM Tris (pH 7.6), 0.3 M KCl, 5 mM
dithiothreitol, and 1 mg/ml BSA. One hundred microliters of
E2 or HPTE ranging in concentration from
10-5–10-10 M were transferred to
a polystyrene tube. [3H]E2
(Amersham Pharmacia Biotech, Arlington Heights, IL) at a
concentration of 5 nM was added to each tube. Recombinant
human ER or ERß (PanVera Corp., Madison, WI) were added at 8 or 11
pmol/ml, respectively, to each tube. Optimal concentrations for each
receptor were empirically determined. After an overnight incubation at
4 C, 100 µl of a 6% hydroxyapatite (HAP) slurry in dilution buffer
[10 mM Tris (pH 7.6) and 5 mM dithiothreitol]
were added to each tube. Tubes were then incubated at 4 C for 30 min
and spun at 1000 x g for 10 min. HAP pellets were
washed four times in dilution buffer containing 1% Triton X-100.
Pellets were resuspended in 1 ml dilution buffer and transferred to
scintillation vials. Radioactivity was measured on a Packard Tri-Carb
460 scintillation counter (Packard Instruments, Meriden, CT).
Statistical analysis
Unless otherwise noted, values presented in this study represent
the mean ± SE 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 Software, Inc., San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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agonist and ERß competitive antagonist or ERß receptor plasmid
along with the estrogen-responsive reporter plasmid C3-Luc (Fig. 1
). HPTE was a complete agonist with both
human and rat ER (Fig. 1, A and C). EC50 values for
induction of luciferase activity for E2 and HPTE
were 4 x 10-9 and 5.1 x 10-8
M, respectively, with human ER and 10-9 and
10-8 M, respectively, with rat ER. Maximal
ER activity was not affected when various concentrations of HPTE
were combined with an inducing concentration of
E2 (10-7 M; Fig. 1, A
and C). In contrast to results obtained with ER, HPTE induced
minimal activity with ERß (Fig. 1, B and D). The maximum activity of
10-5 M HPTE with ERß was only 13% of that
obtained with 10-7 M E2.
In addition, HPTE effectively antagonized
E2-induced ERß activity (Fig. 1, B and D).
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). HPTE caused a parallel shift in the
E2 dose-response curve, indicating that HPTE is
an ERß competitive antagonist. Schild regression analysis yielded a
linear regression with a slope not significantly different from 1 (Fig. 2B). A negative logarithm of the equilibrium dissociation constant
(pKB) value of 7.5 was determined
(KB = 3.05 x 10-8 M)
from the calculated x-intercept in Fig. 2B.
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and ERß (Fig. 3).
The relative binding affinities of ER and ERß for HPTE were 0.004
and 0.02, respectively (E2 = 1.0).
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and ERß in HepG2 and
HeLa cells is not promoter specific and ERß was promoter specific. The
estrogen-responsive MMTV-luciferase reporter (ERE-MMTV-Luc) had high
background activity in HepG2 cells transfected with ER (Fig. 4A). Despite this high background
activity, both E2 and HPTE demonstrated ER
agonist activity. ERE-MMTV-Luc had much lower background in HepG2 cells
when cotransfected with ERß (Fig. 4B), and HPTE exhibited clear ERß
antagonistic activity. HPTE behaved as a partial ER
agonist/antagonist and a complete ERß antagonist in HepG2 (Fig. 4, C
and D) and HeLa (Fig. 4 E and F) cells transfected with the
estrogen-responsive, vitellogenin ERE-luciferase reporter gene
(ERE-Luc; Fig. 4, C and D).
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activity predominates when ER and ERß are
coexpressed and ERß are coexpressed in some tissues in
vivo, and therefore, HepG2 cells were cotransfected with various
concentrations of ER and ERß expression plasmid to determine
overall activity of HPTE when both ER and ERß are present (Fig. 5). A concentration-response curve for
ERß plasmid in the absence of ER is presented in Fig. 5A.
Transfection with increasing concentrations of ERß plasmid into HepG2
cells enhanced peak luciferase activity, but did not alter the overall
agonist-antagonist activity of E2 or HPTE. The
activities of E2 and HPTE in the presence of a
fixed concentration (40 ng/well) of ER plasmid and various amounts
of ERß plasmid are presented in Fig. 5B. Addition of ERß plasmid
had no significant effect on peak E2-induced
luciferase activity, and HPTE behaved primarily as an agonist.
Increasing concentrations of ERß plasmid resulted in decreased
activity of HPTE. However, even when ERß/ER plasmid ratios (by
weight) were 10:1, HPTE still displayed agonist activity. ER
activity also predominated across a complete concentration-response
curve of HPTE when ER and ERß were cotransfected at equal
concentrations (80 ng/well; Fig. 5C). We have confirmed by Western
analysis that ER and ERß are expressed at equivalent
concentrations from their respective expression plasmids under the
conditions of our assay and that transfection with increasing
concentrations of either expression plasmid results in a corresponding
increase in receptor protein (McDonnell, D. P., and J. M.
Hall, unpublished observations).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß. HPTE is an ER agonist, although the
efficacy of the response (complete or partial agonism) depends on the
cell type and promoter. In contrast, HPTE is primarily an ERß
competitive antagonist.
Differential activity of chemicals with ER and ERß has been
previously reported (14, 20, 21, 36, 37). E2
selectively activates ER and inhibits ERß
trans-activation at activating protein-1 sites (21). In
addition, tamoxifen displays some agonist activity with ER, but not
ERß, using ERE-dependent promoters (20). However, this differential
activity of tamoxifen between ER and ERß is highly promoter and
cell specific. Selective action of tamoxifen with ER and ERß may
be due to differences in the activation function (AF1) region, which is
only 30% homologous between the two receptors (14), and previous
studies show that AF1 region is essential for the ER agonist
activity of tamoxifen (32, 35). A recent report using chimeric ER
containing AF2 of ERß and AF1 of ER confirms that differences in
tamoxifen action between ER subtypes are AF1 dependent (36). The roles
of various domains of ER and ERß in the action of HPTE are
currently being investigated. In contrast to the cell-specific
differences observed for tamoxifen as an ER agonist and antagonist, the
differential activity of HPTE with ER and ERß is neither cell type
nor promoter specific. HPTE exhibited primarily ER-dependent agonist
activity and ERß-dependent antagonist activity in HepG2 and HeLa
cells in this study and in three additional human cancer cell lines
derived from three different tissues (data not shown). The
R,R-enantiomer of tetrahydrochrysene has also
recently been shown to have differential ER and ERß activity (37).
Like HPTE, R,R-THC behaves as an ER agonist and an ERß antagonist.
In contrast, the S,S-enantiomer is an agonist
with both ER and ERß. Thus, it is likely that additional chemicals
will be identified that have differential activity at ER and
ERß.
ER and ERß have different, but overlapping, patterns of expression
in vivo. In some tissues, such as the rat uterus, both
receptors are highly expressed (38, 39), whereas in other tissues, such
as rat prostate epithelial cells, only one receptor subtype is
expressed, (40). ER and ERß can form either homodimers or
heterodimers depending on which subtypes are present within the cell
(41, 42, 43, 44). Our results indicate that there is not a simple stoichiometry
when ER and ERß are cotransfected. ER activity predominates in
cotransfections instead of having an intermediate activity between that
demonstrated by either receptor alone. Even a 10-fold excess of
transfected ERß did not significantly diminish activity. Thus, our
results suggest that in tissues where both receptor types are present,
HPTE would predominately act as an ER agonist. However, when only one
receptor subtype is present, then HPTE will primarily act as either an
agonist or an antagonist depending on the receptor subtype present. The
mechanism for this higher than expected activity when both receptor
subtypes are present as well as the tissue selectivity for this
response remain to be determined. Additional studies show that under
some conditions cotransfection of ERß with ER causes a shift to
the right in the dose-response curve (Hall, J. M. and
D. P. McDonnell, unpublished observations). Thus, under these
conditions, low doses that were active with ER alone would not be
active with ER plus ERß.
HPTE is considered the primary metabolite responsible for the
estrogenic effects associated with methoxychlor exposure. Like
E2, methoxychlor is uterotropic in
vivo and induces a number of estrogen-dependent uterine responses
(23, 30, 45, 46, 47). In addition, methoxychlor mimics the action of
E2 on induction of uterine epidermal growth
factors, vaginal estrus, cyclicity, and alterations in sexual behavior
in female rats (24, 48, 49). However, some differences in the in
vivo activity of methoxychlor and E2 have
been reported. Unlike estrogen, methoxychlor does not increase FSH and
LH levels in ovariectomized rats (48). Moreover, methoxychlor acts as
an estrogen agonist in the uterus and an antagonist in the ovary (28).
In addition, dissimilar translation products have been reported in
neonatal mice exposed to E2 or methoxychlor (50, 51). Antagonism of ERß action by HPTE may play a role in responses
induced by methoxychlor that differ from those induced by
E2. For example, the ability of methoxychlor to
act as an antagonist in the ovary may be due to the high level of ERß
expression relative to ER in this tissue (38). HPTE has recently
been shown by us and others to be an androgen receptor antagonist (52, 53), and this may also account for some of the effects associated with
exposure to methoxychlor.
The estimated adult intake of methoxychlor is approximately 0.8
µg/day based on a recent FDA food basket survey that monitored
pesticide residues on food. However, as methoxychlor is rapidly
metabolized and does not bioaccumulate in fat (22), it is unlikely that
HPTE levels would reach the concentrations in humans that would be
sufficient to alter ER, ERß, or androgen receptor activity. HPTE
remains a model xenohormone, however, for several reasons. First, it is
a highly active metabolite of a relatively inactive compound, thus
illustrating the importance of metabolic activation for some
endocrine-active chemicals. Second, the physiological consequences of a
chemical that is a relatively high affinity ER agonist, ERß
antagonist, and androgen receptor antagonist are unknown, and HPTE can
serve as a model for investigating the in vivo effects of an
agent that modulates multiple endocrine pathways. Finally, our current
studies with HPTE as well as our recent publication demonstrating the
unique estrogenic activity of bisphenol A (33) illustrate the
difficulty in labeling a chemical as an estrogen receptor agonist or an
androgen receptor antagonist. As our current understanding of steroid
hormone receptor function evolves, it is likely that other chemicals
will be identified as selective hormone receptor modulators with a
broad spectrum of activities that differ from endogenous hormones
steroids, and therefore, a more detailed understanding of their
mechanism of action will be required. Additional studies with HPTE and
structural analogs may lead to further insights on ligand specificity
for ER and ERß action and to a better understanding of the
physiological roles of these two receptors.
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Acknowledgments |
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Footnotes |
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Received April 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in vivo and in vitro. Biochem Biophys Res Commun 243:122–126[CrossRef][Medline]
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receptor chimeras. Endocrinology 139:4513–4522 or
estrogen receptor ß. Endocrinology 140:800–804 and ß form heterodimers on DNA. J Biol
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F. Larrea, R. Garcia-Becerra, A. E. Lemus, G. A. Garcia, G. Perez-Palacios, K. J. Jackson, K. M. Coleman, R. Dace, C. L. Smith, and A. J. Cooney A-Ring Reduced Metabolites of 19-nor Synthetic Progestins as Subtype Selective Agonists for ER{alpha} Endocrinology, September 1, 2001; 142(9): 3791 - 3799. [Abstract] [Full Text] [PDF]
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 K. S. Bramlett, Y. Wu, and T. P. Burris Ligands Specify Coactivator Nuclear Receptor (NR) Box Affinity for Estrogen Receptor Subtypes Mol. Endocrinol., June 1, 2001; 15(6): 909 - 922. [Abstract] [Full Text] [PDF]
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H. Tamura, S. C. Maness, K. Reischmann, D. C. Dorman, L. E. Gray, and K. W. Gaido Androgen Receptor Antagonism by the Organophosphate Insecticide Fenitrothion Toxicol. Sci., March 1, 2001; 60(1): 56 - 62. [Abstract] [Full Text] [PDF]
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 K. W. Gaido, S. C. Maness, D. P. McDonnell, S. S. Dehal, D. Kupfer, and S. Safe Interaction of Methoxychlor and Related Compounds with Estrogen Receptor alpha and beta , and Androgen Receptor: Structure-Activity Studies Mol. Pharmacol., October 1, 2000; 58(4): 852 - 858. [Abstract] [Full Text]
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 M. R. Walters, M. Dutertre, and C. L. Smith SKF-82958 Is a Subtype-selective Estrogen Receptor-alpha (ERalpha ) Agonist That Induces Functional Interactions between ERalpha and AP-1 J. Biol. Chem., January 11, 2002; 277(3): 1669 - 1679. [Abstract] [Full Text] [PDF]
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) or HPTE (
) or with HPTE plus
10 nM E2 (
). After 24-h incubation, cultures
were assayed for both luciferase and ß-galactosidase activities.
Luciferase activity was normalized to ß-galactosidase activity.
Values represent the mean ± SE of three or four
separate experiments and are presented as the percent response, with
100% activity defined as the activity achieved with 10-7
M E2.
), and 10-5 M HPTE (
)
in HepG2 cells transfected with expression plasmids for human ERß,
C3-Luc, and ß-galactosidase. Values represent the mean ±
SE normalized luciferase activity from three separate
assays. B, Schild regression analysis of the data shown in A. The dose
ratio (dr) is [A'] ÷ [A], where [A'] and [A] refer to
equiactive concentrations of E2 in the presence and absence
of HPTE, respectively.
