Endocrine Disrupting Chemicals Targeting Estrogen Receptor
Signaling: Identification and Mechanisms of Action
Erin K. Shanle‡
and Wei Xu*,†,‡
McArdle Laboratory for Cancer Research, UniVersity of Wisconsin, 1400 UniVersity AVenue, Madison,
Wisconsin 53706, United States, and Molecular and EnVironmental Toxicology Center, UniVersity of Wisconsin,
Madison, Wisconsin 53706, United States
ReceiVed July 8, 2010
Many endocrine disrupting chemicals (EDCs) adversely impact estrogen signaling by interacting
with two estrogen receptors (ERs): ERR and ERβ. Though the receptors have similar ligand binding
and DNA binding domains, ERR and ERβ have some unique properties in terms of ligand selectivity
and target gene regulation. EDCs that target ER signaling can modify genomic and nongenomic ER
activity through direct interactions with ERs, indirectly through transcription factors such as the
aryl hydrocarbon receptor (AhR), or through modulation of metabolic enzymes that are critical for
normal estrogen synthesis and metabolism. Many EDCs act through multiple mechanisms as
exemplified by chemicals that bind both AhR and ER, such as 3-methylcholanthrene. Other EDCs
that target ER signaling include phytoestrogens, bisphenolics, and organochlorine pesticides, and
many alter normal ER signaling through multiple mechanisms. EDCs can also display tissue-selective
ER agonist and antagonist activities similar to selective estrogen receptor modulators (SERMs)
designed for pharmaceutical use. Thus, biological effects of EDCs need to be carefully interpreted
because EDCs can act through complex tissue-selective modulation of ERs and other signaling
pathways in ViVo. Current requirements by the U.S. Environmental Protection Agency require some
in Vitro and cell-based assays to identify EDCs that target ER signaling through direct and metabolic
mechanisms. Additional assays may be useful screens for identifying EDCs that act through alternative
mechanisms prior to further in ViVo study.
Contents
1. Introduction 6
2. Estrogen Receptor Signaling 7
2.1. ERR and ERβ Subtypes 7
2.2. ER Signaling Pathways 8
2.3. Selective Estrogen Receptor Modulators
(SERMs) and EDCs
8
3. Direct Action of EDCs on ER Signaling 9
3.1. Structural Features of Estrogenic Chemicals 9
3.2. Known Estrogenic EDCs 9
3.2.1. High Affinity Pharmaceuticals: DES 9
3.2.2. Organochlorine Pesticides: Methoxychlor
and DDT
10
3.2.3. Industrial Phenolics: BPA and
Alkylphenols
11
3.2.4. Phytoestrogens: Liquiritigenin and
Genistein
11
4. Indirect Actions of EDCs on ER Signaling 12
4.1. Aryl Hydrocarbon Receptor Ligands and
Functional Cross-Talk with ERs
12
4.2. Metabolic Modulators of Estrogen Metabolism 13
5. In Vitro and Cell Based Approaches to Identify
EDCs That Impact ER Signaling
13
5.1. Assays to Measure Binding and Dimerization 14
5.2. Transcriptional Assays 14
5.3. Proliferation Assays 14
5.4. Metabolic Assays 14
6. Conclusions and Future Perspectives 15
1. Introduction
Endocrine disrupting chemicals (EDCs1
) are compounds in
the environment or diet that interfere with normal hormone
biosynthesis, signaling, or metabolism (1). Many EDCs display
estrogenic activity and interfere with normal estrogen signaling,
which is mediated by two estrogen receptors (ERs): ERR and
ERβ. ERR and ERβ have both unique and overlapping physiological
roles in mediating estrogen signaling which are
dependent on the cellular context, the availability of cofactors,
and the ligand. EDCs that target ER signaling can modify
genomic and nongenomic ER activity through direct interactions
with ERs, indirectly through transcription factors such as the
aryl hydrocarbon receptor (AhR), or through modulation of
metabolic enzymes that are critical for normal estrogen synthesis
* To whom correspondence should be addressed. Tel: 608-265-5540.
Fax: 608-262-2824. E-mail: wxu@oncology.wisc.edu.
†
McArdle Laboratory for Cancer Research.
‡
Molecular and Environmental Toxicology Center.
1
Abbreviations: 3-MC, 3-methylcholanthrene; AF, activation function;
AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear
translocator; bHLH, basic-helix-loop-helix; BPA, bisphenol A; BRET,
bioluminescence resonance energy transfer; CYP, cytochrome P450dependent
monooxygenase; DBD, DNA binding domain; DDT, 1,1,1trichloro-2,2-bis(p-chlorophenyl)ethane;
DES, diethylstilbestrol; DPN, diarylpropionitrile;
E2, 17β-estradiol; EDC, endocrine disrupting chemical;
ER, estrogen receptor; ERE, estrogen response element; FRET, fluorescence
resonance energy transfer; FSH, follicle stimulating hormone; HAH,
halogenated aromatic hydrocarbon; HDX, hydrogen/deuterium exchange;
HPTE, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane; LH, leutinizing
hormone; LBD, ligand binding domain; NR, nuclear receptor; PAH,
polycyclic aromatic hydrocarbon; PAS, PER-ARNT-SIM; RBA, relative
binding affinity; SERM, selective estrogen receptor modulator; TCDD,
2,3,7,8-tetrachlorodibenzo-p-dioxin.
Chem. Res. Toxicol. 2011, 24, 6–196
10.1021/tx100231n  2011 American Chemical Society
Published on Web 11/05/2010
and metabolism. Though the receptors bind endogenous estrogen,
17β-estradiol (E2), with similar affinities, many exogenous
ligands have been shown to display selectivity for ERR or ERβ
(2). ERs have relatively large ligand binding pockets and are
relatively promiscuous nuclear receptors in terms of binding
exogenous chemicals. Despite broad specificity for ligands, both
ER subtypes have ligand binding pockets that determine
common structural features of estrogenic ligands. Though EDCs
function through multiple mechanisms, many display impacts
on ER signaling by directly binding with the ER ligand binding
pocket. Such direct acting EDCs include pharmaceutical chemicals,
bisphenols, phytoestrogens, and organochlorine pesticides.
Often, EDCs act through multiple mechanisms, including
indirect action through activation of other transcription factors,
most notably the aryl hydrocarbon receptor, or through modification
of estrogen metabolism.
In Vitro and cell-based assays for detecting estrogenic EDCs
are valuable because they are targeted tests that can often be
performed in a high-throughput manner with lower costs than
in ViVo animal studies. Current guidelines for detecting direct
acting estrogenic EDCs in Vitro are aimed at identifying
chemicals that bind ER and initiate the transcription of ER
targets. Several additional high-throughput strategies are available
to identify EDCs that target ERs, but most are limited to
the identification of direct acting EDCs. Transcriptional reporter
assays, bioluminescence or fluorescence resonance energy
transfer (BRET or FRET), and fluorescence polarization assays
can be used to successfully identify chemicals that directly
interact with ERs. It is much more difficult to identify EDCs
that interfere with normal ER signaling indirectly, but in Vitro
and cell-based assays aimed at identifying chemicals that
interfere with estrogen metabolism are currently available. As
our understanding of ER signaling expands, the development
of targeted in Vitro assays will aid in the identification of EDCs
prior to further in ViVo study.
2. Estrogen Receptor Signaling
Two estrogen receptors, ERR and ERβ, regulate gene
expression in response to estrogen exposure. ER signaling may
occur in a ligand dependent or ligand independent manner.
Estrogen target genes are regulated through genomic pathways,
either by direct ER-DNA interactions or through a tethering
mechanism in which ERs tether to DNA through other
transcription factors, and nongenomic pathways in which
estrogen exposure leads to rapid activation of kinase signaling
cascades. The multiple signaling pathways mediated by the two
ER subtypes complicate the effects of EDCs on estrogen
signaling, and each subtype may mediate unique responses to
ligands. Importantly, the tissue specific effects of ER signaling
are complex and EDCs can display selective modulation of ER
signaling.
2.1. ERr and ERβ Subtypes. For decades, ERR was thought
to be the only ER regulating estrogen signaling, and the vast
majority of studies regarding the effects of EDCs have been
conducted with a focus on ERR. In 1996, a second ER, ERβ,
was cloned from a rat prostate and ovary, thereby shifting our
understanding of ER signaling (3). ERR and ERβ are encoded
by distinct genes on separate chromosomes (4) and display
unique and overlapping physiological roles that are highly
dependent on the tissue and cell type. ERs mediate estrogen
signaling in reproductive tissues and nonreproductive tissues
including the brain, lungs, colon, prostate, and cardiovascular
system. Both receptors are expressed in the brain, lung, uterus,
breast, heart, and intestine. ERR shows unique expression in
hepatocytes and the hippocampus, while ERβ is the prominent
subtype expressed in the prostate, vagina, and cerebellum (5).
Based on observations of ERR and ERβ knockout mice, ERR
is the dominant receptor regulating normal mammary development,
while ERβ is less critical for normal mammary and
reproductive development (6, 7). ERβ is thought to oppose the
proliferative action of ERR in mammary cells (8-11), but the
physiological role of ERβ in many tissues is still under debate.
The structural features of ERR and ERβ have been reviewed
extensively (12, 13); therefore, the discussion of ERR and ERβ
is limited to key differences within the activation function (AF)
regions and ligand binding and DNA binding domains of the
receptors. Two activation functions, AF-1 and AF-2, interact
with cofactors to down- or up-regulate the transcription of target
genes. The N terminal AF-1 domain can regulate transcription
independent of the ligand, while AF-2, found in the ligand
binding domain (LBD), regulates transcription in a ligand
dependent manner. ERR and ERβ have strikingly different AF-1
domains; they share only 18% similarity, and the AF-1 domain
of ERR enhances estrogen-induced expression of reporter genes
to a greater extent than that of ERβ (14, 15). The AF-2 domain
consists of four R-helices (H3, H4, H5, and H12) that form a
hydrophobic groove to which cofactors with an LXXLL motif,
known as a nuclear receptor (NR) box, can bind. The LBDs of
ERR and ERβ share 59% similarity, but the receptors only differ
in two amino acids in the ligand binding pocket: Leu 384 of
ERR corresponds to Met 336 of ERβ, and Met421 of ERR
corresponds to Ile373 of ERβ. Such slight differences within
the ligand binding pockets contribute subtype selectivity to some
chemicals though the receptors bind E2 with similar affinities
(2). The DNA binding domains (DBDs) of ERR and ERβ share
97% similarity, and the receptors bind DNA at estrogen response
elements (EREs), 13 base pair inverted repeats (GGTCAnnnTGACC),
to regulate target gene expression. Many ER target
genes do not contain perfect EREs in their promoters, though
chromatin immunoprecipitation coupled with gene expression
microarrays has shown ERR and ERβ genomic binding sites
often contain ERE-like sequences. In MCF7 breast cancer cells,
71% of ERR binding sites contained full EREs with at most 2
base pair deviations from the consensus ERE sequence; 25%
of the binding sites contained half ERE sequences (16). The
majority of binding sites occurred in distal regions or intragenic
regions, and only 5% of binding sites fall within the proximal
promoter regions of target genes (16, 17). Genomic mapping
of ERβ binding sites in MCF7 cells in which ERβ was inducibly
overexpressed revealed that ERβ binding regions mapped more
closely to transcription start sites compared to those of ERR
(18). Of all ERβ binding sites, only 5% contained an isolated
ERE or ERE-like site, and approximately 60% of binding sites
contained ERE-like sequences and activator protein 1 (AP-1)
binding sequences. In a comparison of the ERR and ERβ binding
sites observed in MCF7 cells, over 75% of ERβ binding sites
were also identified as ERR binding sites (19). Since the DNA
binding domains of ERR and ERβ display high homology and
there is significant overlap in their genomic binding sites, it is
not surprising that these receptors can regulate common targets;
however, unique gene regulation has been observed in U2OS
osteosarcoma cells and Hs578T breast cancer cells engineered
to express ERR or ERβ (20, 21). It should be noted that ERR
and ERβ form both homodimers and heterodimers in response
to ligand binding and, like ER homodimers, heterodimers are
capable of binding EREs (22, 23). The biological role of ER
heterodimers is still unclear, and thus, the impacts of EDCs on
heterodimer activity are largely unknown.
ReView Chem. Res. Toxicol., Vol. 24, No. 1, 2011 7
2.2. ER Signaling Pathways. Estrogen signaling occurs
through multiple pathways in which ERs regulate the transcription
of target genes directly or indirectly. Ultimately, the impacts
of EDCs on ER signaling are best understood in a ligand
dependent context. Inappropriate ER signaling can lead to
increased risk of hormone dependent cancer, impaired fertility,
abnormal fetal growth and development, and altered metabolism
in white adipose tissue (1). The effects of EDCs are not limited
to the ligand dependent activity of ERs, but it is the best studied
aspect of ER targeted endocrine disruption. The mechanisms
of ligand independent ER signaling are complex and involve
the activation of multiple signaling pathways (13). The discussion
of ER signaling mechanisms herein is limited to the ligand
dependent activation pathways.
ERs signal through both genomic (nuclear) and nongenomic
(extra-nuclear) pathways in response to the ligand (Scheme 1).
The mechanisms of ER signaling have been discussed extensively
in the literature (12, 13), and genomic and nongenomic
ER signaling mechanisms are presented here in brief. In the
genomic pathway, ERs mediate target gene regulation through
binding directly to EREs or tethering to EREs through transcription
factors such as specificity protein 1 (Sp1) or AP-1 at their
cognate response elements (24, 25). In classical genomic
signaling, ligands bind the receptor in the LBD which induces
conformational changes allowing dimerization and DNA binding.
Crystal structures of the LBD of ERR and ERβ bound to
agonists, compounds that lead to transcriptional activation, or
antagonists, compounds that inhibit transcriptional activation,
reveal similar structural features of ERR and ERβ that contribute
to cofactor recruitment and transcriptional output (26-28).
When bound to agonists such as the endogenous estrogen E2,
conformational changes in the receptors reveal a cofactor
binding site. Ligands may induce conformations that preferentially
recruit specific cofactors thereby inducing differential
responses. The best described nuclear receptor cofactors are the
p160 family of coactivators, namely, SRC-1, SRC-2, and SRC-
3, but the cofactor complexes that mediate the ultimate outcome
of ER signaling are complicated; more that 300 cofactors have
been described in the literature (29).
The nongenomic pathway involves the activation of other
signal transduction pathways that lead to rapid responses, within
minutes, to estrogen exposure. The mechanism of nongenomic
ER signaling is not clear and is thought to be mediated by
membrane associated full length ERs or a receptor distinct from
ERR and ERβ (30). A G-protein coupled receptor known as
GPR30 mediates rapid estrogen signaling independent of ERs
(31), which can lead to activation of the MAPK or PI3 kinase
signaling cascades (32, 33), fluctuations in intracellular calcium
(34), or stimulation of cAMP production (35). Known EDCs
such as bisphenol A (BPA) and diethylstilbestrol (DES) also
induce rapid estrogen signaling (36), indicating that EDCs can
exert effects on nongenomic ER signaling.
As mentioned previously, ER signaling can occur nonclassically
through AP-1 and Sp1 transcription factors in response
to estrogenic ligands including known EDCs. The mechanisms
of ER/AP-1 and ER/Sp1 mediated transactivation of target genes
are complex and depend on ERR or ERβ subtype, cell context,
and the type of ligand (37). Transient transfections with a
reporter construct containing a GC-rich Sp1 binding site in the
promoter demonstrated that ERR can mediate estrogen responsive
reporter expression in breast and prostate cancer cells but
not in HeLa cervical cancer cells (25). Though ICI 182,780 and
tamoxifen are considered antiestrogenic ligands, treatments with
either compound led to transcriptional activation of the Sp1
reporter similar to E2, demonstrating the importance of promoter
context in the expression of target genes in response to ligand
binding. Transcription of the Sp1 reporter construct in response
to E2 treatment did not occur in cells transfected with ERβ,
indicating that ER subtypes display unique actions through
classical Sp1 binding sites. EDCs can also induce ERR/Sp1
transactivation. In MCF7 cells transfected with wild-type ERR
and a Sp1 reporter construct, known EDCs including octylphenol,
nonylphenol, BPA, and other xenoestrogens activated the
expression of the Sp1 reporter similar to E2 (38).
Some EDCs could potentially display pathway selective
actions. For example, WAY-169916 is a nonsteroidal selective
inhibitor of NFκB activity that works in an ER dependent
manner (39). ERR and ERβ activation can antagonize the
functional activity of NFκB in response to estrogen treatment
in a wide variety of cell lines (reviewed in ref 40). WAY-169916
is unique in that the compound inhibits NFκB action only in
the presence of ERR or ERβ and is devoid of classical estrogenic
activity as it does not induce the expression of ERE reporter
constructs in the presence of ERR or ERβ. The pathway
selective inhibitory effects of WAY-169916 are maintained in
ViVo as measured by a reduction in inflammatory gene expression
in the absence of classical estrogenic activity including
uterotrophic side effects (39). The pathway selective action of
WAY-169916 demonstrates the potential for EDCs to elicit very
specific effects mediated by ERs in the absence of classical ER
signaling.
2.3. Selective Estrogen Receptor Modulators (SERMs)
and EDCs. For decades, the pharmaceutical industry has worked
to develop SERMs that act as agonists and antagonists in a tissue
selective manner. In the context of EDCs, SERMs provide a
useful framework for understanding specific signaling elicited
by ER ligands. The SERM tamoxifen has been used as an
effective therapy for treating ERR positive breast cancers for
the past 30 years and has contributed to a decline in breast cancer
mortality rates (41). Tamoxifen antagonizes ERR in mammary
cells, thereby inhibiting ERR mediated breast cancer progression
Scheme 1. Ligand Dependent ER Signaling Pathwaysa
a
Genomic signaling occurs when ligands enter the cell and bind ER to
induce dimerization. ER dimers bind DNA directly at ERE sequences or
indirectly by tethering to DNA through other transcriptions factors such as
Sp1 or AP-1. Nongenomic signaling occurs when ligands bind membrane
bound receptors, which leads to the activation of kinase signaling cascades.
8 Chem. Res. Toxicol., Vol. 24, No. 1, 2011 Shanle and Xu
and is an ERR agonist in the endometrium which contributes
to increased risk for endometrial cancers after extended exposure
(41). The tissue specific effects of tamoxifen are due in part to
coactivator availability and cell context. In Ishikawa endometrial
cells, tamoxifen induces cell cycle progression, and the proliferative
effect of tamoxifen is lost when SRC-1 expression is
knocked down (42). SRC-1 expression is higher in Ishikawa
cells compared to that in MCF7 breast cancer cells, a trend
observed across a number of endometrial and breast cancer cell
lines, suggesting that SRC-1 may mediate some of the tissue
specific effects of tamoxifen.
In Vitro assays for investigating SERMs have been extensively
applied to EDCs, and the results suggest that many EDCs are
SERMs. For example, experiments with ER-negative MDAMB-231
breast cancer cells transfected with wild-type or a
truncated ERR variant that cannot recruit cofactors through AF-2
demonstrate the SERM-like properties of some EDCs (43). In
cells transfected with wild-type ERR, treatment with E2,
tamoxifen, and xenoestrogens including BPA, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane
(HPTE), and nonylphenol
induce expression of an ERE-luciferase reporter demonstrating
the estrogenic activity of all the ligands. In cells transfected
with truncated ERR, E2 and the alkylphenols could not activate
the expression of the reporter while tamoxifen, BPA, and HPTE
were able to activate ER mediated luciferase expression indicating
BPA and HPTE have SERM-like properties similar to those
of tamoxifen. Pharmaceutical SERMs demonstrate that estrogenic
ligands can modulate ER signaling in a tissue selective
manner and that EDCs can have similar selective properties.
3. Direct Action of EDCs on ER Signaling
Many EDCs directly impact ER signaling by competing with
endogenous estrogen for ER binding sites. EDCs that show
estrogenic activity through ER binding may have structural
features shared with E2 that are determined by the ligand binding
pockets of ERR and ERβ. Though ERR and ERβ have very
similar ligand binding pockets, subtle differences in the amino
acids that line the pocket contribute to ligand selectivity between
the subtypes (44). EDCs that display direct impacts on ER
signaling through interaction with ER LBDs include pharmaceutical
chemicals, industrial bisphenolics, organochlorine
pesticides, and phytoestrogens.
3.1. Structural Features of Estrogenic Chemicals. Crystal
structures of the LBDs of ERR and ERβ reveal features of the
ligand binding pockets that are critical for understanding which
compounds may display estrogenic activity through direct
interaction with ERR or ERβ. ERs are fairly promiscuous
nuclear receptors in terms of ligand selectivity; the ligand
binding pockets of ERR and ERβ are significantly larger, 450
and 390 Å3
, respectively, than E2, just 245 Å3
, allowing a
diverse set of small molecules access to the LBD (26, 28). Both
receptors display similarly high affinities for E2 due to
hydrophobic interactions and a network of hydrogen bonds
between the hydroxyl groups on E2, a water molecule, and
amino acids that line the ligand binding pocket. Glu353 and
Arg394 of ERR and Glu305 and Arg346 of ERβ share hydrogen
bonds with a water molecule and the hydroxyl in the A ring of
E2. On the other side of the E2 molecule, the hydroxyl of the
D ring shares a hydrogen bond with His524 of ERR, corresponding
to His475 in ERβ (Figure 1A). Ligands that bind ERs
directly in the LBD share structural similarities with E2 in that
they typically have hydroxyls that undergo hydrogen bonding
with the Glu, Arg, and His residues in the ligand binding pocket.
Though ERR and ERβ have similar affinities for E2, there are
many ligands that display selectivity for ERR or ERβ, including
known EDCs. Differences in two amino acids within the ligand
binding pocket are the major determinants of the subtype
selectivity of some ligands. In helix 5, Leu384 of ERR
corresponds to Met336 of ERβ, and in loop 6-7, Met421 of
ERR corresponds to Ile373 of ERβ (Figure 1B). In light of the
potential antiproliferative role of ERβ, significant efforts have
focused on developing ERβ selective ligands. On the basis of
the structural similarities of known ERβ selective compounds,
structural determinants of ERβ selectivity and binding affinity
can be useful for predicting chemicals that will directly bind
ERs and potentially display subtype selectivity (45). Binding
affinities for the receptors are often expressed relative to E2,
and the relative binding affinities (RBAs) of many estrogenic
ligands have been characterized (2, 44).
3.2. Known Estrogenic EDCs. EDCs can impact estrogen
signaling through interaction with the LBDs of ERR and ERβ,
thereby activating the receptors inappropriately. Examples of
such direct acting EDCs include pharmaceuticals, phytoestrogens,
pesticides, and industrial chemicals. Here, we highlight
known EDCs that impact ER signaling in part through interaction
with the ligand binding pockets (Figure 2). It must be noted
that EDCs often act through multiple mechanisms and can
display tissue specific effects. Phytoestrogens can bind ERs with
relatively low affinities but display ERβ selectivity, suggesting
that EDCs may have impacts on ER subtype specific signaling
pathways. Other estrogenic EDCs, such as the pharmaceutical
DES, can interact with ligand binding pockets of ERs with very
high affinity and impact both genomic and nongenomic ER
signaling. The complex responses to EDCs such as DES
highlight the complexity of predicting the effects of EDCs on
the multiple signaling pathways mediated by ERs. This review
will provide examples of known EDCs, and additional recent
reviews have more comprehensive information regarding EDCs
(1, 46-48).
3.2.1. High Affinity Pharmaceuticals: DES. One of the first
examples of endocrine disruption occurred when women were
exposed to DES (Figure 2, 2) as a therapy for preventing
miscarriages during pregnancy; prenatal exposure to DES was
later linked to vaginal cancer in daughters of mothers taking
DES (49) and structural, functional, and cellular abnormalities
in the reproductive system of males exposed to DES in utero
(50). DES has been a model for EDC action and has been
extensively studied because of its significant adverse impacts
Figure 1. Crystal structures of ERR and ERβ LBD bound to E2 (shown
in red) modified from PDB 1ERE and PDB 2J7X. (A) Comparison of
the ERR and ERβ hydrogen bond network with E2 (H bonds shown in
green); (B) ERR and ERβ differ in only two amino acids in the ligand
binding pocket (shown in yellow).
ReView Chem. Res. Toxicol., Vol. 24, No. 1, 2011 9
on humans in utero (51). DES appears to act through both
genomic and nongenomic ER signaling to induce adverse ER
signaling. It is structurally quite similar to E2, and crystal
structures of ER LBD bound to DES show the hydroxyl groups
of DES are similarly positioned as those of E2. DES displays
even higher affinity for ERs due to additional hydrophobic
interactions that further stabilize the ligand (2, 27). Because it
has high affinity for the receptors, it is a potent transcriptional
activator through genomic signaling. High incidence of uterine
tumors occurs in mice after neonatal treatment with DES, and
similar effects are observed for other estrogenic ligands including
E2 and genistein (51). More recent evidence suggests DES
can impact nongenomic estrogen signaling as well (36, 52). DES
treatment of MCF7 breast cancer cells leads to rapid activation
of PI3 kinase signaling and phosphorylation of AKT. Furthermore,
activation of the signaling cascade leads to phosphorylation
of EZH2, a histone methyl transferase, and modification
of the chromatin structure, which may contribute to the
epigenetic effects of DES (52).
3.2.2. Organochlorine Pesticides: Methoxychlor and DDT.
Methoxychlor and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane
(DDT) are organochlorine pesticides that can exhibit estrogenic
activity through interaction with ERR and ERβ LBDs (44). DDT
and methoxychlor adversely affect the female reproductive tract
by stimulating uterine proliferation and impairing normal follicle
development (reviewed in ref 53). Though DDT was banned
in the 1970s, it remains an EDC of interest due to its persistence
in the environment and its accumulation in adipose tissue. DDT
and the dechlorination metabolite dichlorodiphenyldichloroethylene
(DDE) have been detected in the adipose tissue of
humans throughout the world (54). DDT occurs as a mixture
of three isomers: p,p′-DDT, o,p′-DDT, and o,o′-DDT. The
estrogenicity of DDT arises primarily from o,p′-DDT (Figure
2, 3), which can bind ERR and ERβ with RBAs of 0.01 and
0.02, respectively; p,p′-DDT (Figure 2, 4) has RBAs <0.01 for
both receptors (44).
Methoxychlor (Figure 2, 5) was developed as an alternative
to DDT, but its use was also banned by the United States
Environmental Protection Agency in 2003 due to its endocrine
disrupting properties (55). Methoxychlor induces a variety of
adverse physiological effects (reviewed in ref 56), but most
notably, it stimulates uterotrophic activity and impairs overall
fertility in rat models. Though methoxychlor has relatively low
binding affinities for ERR and ERβ (RBAs of <0.01 for ERR
and ERβ (44)), the major metabolite of methoxychlor, HPTE
(Figure 2, 6), exhibits unique estrogenic activity, which likely
mediates the endocrine disrupting properties of methoxychlor.
HPTE can act as an agonist for ERR and an antagonist for ERβ
(57). In HepG2 liver carcinoma cells transfected with ERR or
ERβ and a luciferase reporter linked to an estrogen responsive
complement 3 promoter, HPTE induced maximal reporter
expression relative to E2 only in cells expressing ERR. In cells
transfected with ERβ, maximal HPTE-induced luciferase expression
only reached 13% that of the maximal E2 response,
indicating its agonistic properties are ERR selective. HPTE
selectively antagonizes E2 mediated ERβ activation. HPTE
cotreatment antagonized E2 induction of luciferase in HepG2
cells transfected with ERβ but had no effect on E2 induced
luciferase expression in cells transfected with ERR (57). ERR
selective agonistic properties were also observed for similar
chemicals with bishydroxyphenyl core structures indicating that
many structurally related compounds may impact ER signaling
primarily through ERR (58). HPTE may also mediate effects
through nonclassical ER signaling mechanisms similar to those
Figure 2. Chemical structures of estrogenic ligands and EDCs that target ER signaling.
10 Chem. Res. Toxicol., Vol. 24, No. 1, 2011 Shanle and Xu
of E2. HPTE and E2 treatment led to similar gene expression
profiles in utero from mice expressing an ERR mutant deficient
in DNA binding, which limits ERR mediated gene regulation
to the pathway in which ERR tethers to DNA through transcription
factors such as AP-1 and Sp1 (59). Additionally, ERR
knockout mice did not respond to HPTE treatment, indicating
the effects of HPTE on gene regulation in the mouse uterus are
dependent on ERR.
3.2.3. Industrial Phenolics: BPA and Alkylphenols. Chemicals
used for industrial purposes are often used in large
quantities, and exposure risks are high due to the large volume
and application of such chemicals. BPA (Figure 2, 7) is a
monomer of polycarbonate plastics that is one of the highest
volume chemicals used in industry. Exposure to BPA is thought
to occur primarily through ingestion since polycarbonate plastics
are used in food and water containers, though it is also present
in medical tubing and epoxy resin. A recent study by the Center
for Disease Control estimates that over 90% of the United States
population have significant levels of BPA in the urine (60). The
estrogenic effects of BPA have been demonstrated with in Vitro
and in ViVo experiments, but there is controversy surrounding
the potential for BPA to significantly impact normal endocrine
function (reviewed in ref 61). BPA has two phenolic rings,
similar to DES, but displays much lower binding affinities and
transcriptional potencies for ERR and ERβ compared to those
of DES or E2 (RBA of 0.01 for ERR and ERβ) (44, 62). BPA
can induce a uterotrophic response in immature CD-1 mice after
3 days of exposure but only at high doses (100 mg/kg) (63);
human daily intake is estimated to be around 1 µg/kg (61). BPA
can also elicit signaling through nonclassic ER signaling
pathways at high concentrations. In mice expressing a mutant
form of ERR that is deficient in DNA binding which limits ER
mediated responses to pathways in which ERR is tethered to
DNA through other transcription factors, relatively high doses
of BPA elicit uterine gene expression profiles similar to those
of E2 (59). Despite the evidence that BPA elicits estrogenic
responses in many experimental systems, there is uncertainty
as to whether such evidence can be extrapolated to human
exposure levels considering the relatively high doses of BPA
required to elicit such responses.
Given the low affinity of BPA for ERs, it is likely that the
estrogenic effects of BPA are due to nongenomic ER signaling.
BPA can bind membrane associated ERR or GPR30 and initiate
rapid signaling (64, 65). BPA has higher affinity for GPR30
compared to that of ERR and ERβ (RBA for GPR30 of 2.83)
(65). Nanomolar doses of BPA stimulated a calcium flux in
GH3/B6 pituitary cells in which ERR is membrane associated,
indicating that rapid signaling can be triggered at environmentally
relevant concentrations of BPA (64). At higher concentrations
of BPA, in the micromolar range, nongenomic signaling
is activated in MCF7 cells as indicated by the detection of
phosphorylated AKT and phosphorylated ERK proteins and
expression of transiently transfected reporter constructs specifically
responsive to MAPK and PI3K activation (66). The ability
of BPA to induce genomic ER mediated transcription is
dependent on the availability of cofactors. In HeLa cells
transfected with ERR or ERβ and the coactivators TIF2 or SRC-
1, BPA had greater effects on gene expression in cells expressing
ERβ when TIF-2 was the available cofactor, but similar effects
in cells expressing ERR or ERβ when SRC-1 was present (67).
The complex mechanisms through which BPA can initiate
signaling contribute to the controversy surrounding BPA and
its role as an EDC.
Other industrial chemicals that can directly interact with ER
LBDs and elicit estrogenic responses include alkyl phenols such
as 4-t-octylphenol (Figure 2, 8) and nonylphenol (Figure 2, 9)
(44, 68). Nonylphenol has a relatively weak affinity for ERs
(RBAs for ERR and ERβ of 0.05 and 0.09, respectively) as
does 4-tert-octylphenol (RBAs for ERR and ERβ of 0.02 and
0.07, respectively) (44), but alkylphenols still induce estrogenic
responses. In a comparison of gene expression profiles in MCF7
breast cancer cells after treatment with E2 or a variety of
alkylphenols, a high correlation was observed in profiles from
cells treated with E2 and nonylphenol or 4-tert-octylphenol (69).
Like BPA, nonylphenol also shows a higher binding affinity
for GPR30 compared to that of ERR and ERβ (RBA of 2.15)
(65) and can induce nongenomic responses such as ERK
phosphorylation and rapid calcium flux through membrane
associated ERR (70). Comparisons of the effects elicited by
alkylphenols and the hydrophobicity of the chemicals revealed
that more hydrophobic alkylphenols such as nonylphenol and
4-tert-octylphenol were more potent activators of calcium flux
and less potent inducers of ERK phosphorylation in GH3/B6/
F10 pituitary tumor cells (70).
3.2.4. Phytoestrogens: Liquiritigenin and Genistein. Plants
produce a wide variety of secondary metabolites, some of which
are phytoestrogens, chemicals that have structures similar to
that of E2 and display estrogenic activity through ER signaling
pathways. Phytoestrogens have poly phenolic structures and can
be classified as flavonoids, coumestans, and lignans (44).
Exposure occurs primarily through dietary intake of beverages
and foods containing fruits, herbs, and vegetables, most notably
soy, which have high levels of phytoestrogens (71). Flavonoids
are one of the most prevalent classes of phytoestrogens found
in dietary sources and are further classified as chalcones,
flavones, flavonols, flavanones, flavanols, anthocyanins, and
isoflavones. Most studies have focused on resveratrol, quercetin,
daidzein, and genistein as they are some of the most commonly
ingested phytoestrogens (72).
Though phytoestrogens are often discussed in the context of
cancer prevention and do not typically elicit physiological
abnormalities such as DES or other environmental EDCs,
phytoestrogens cannot be neglected as EDCs due to widespread
exposure to plant material in the diet and the profound
physiological effects mediated by ERs (72). Daily phytoestrogen
intake ranges from 0.15-3 mg per day in the United States
(72-74). Phytoestrogens have relatively weak affinities for ERR
and ERβ, but serum levels can reach near micromolar concentrations
after a soy-rich meal (75), well above the concentration
of endogenously circulating estrogens (20-200 pg/mL). Phytoestrogens
can act as endocrine disruptors by affecting estrogen
biosynthesis and the menstrual cycle (72). Menstrual cycles were
longer in women consuming 40 mg of isoflavones per day for
3 months, indicating that phytoestrogen can perturb the normal
estrous cycle (76). Phytoestrogen exposure in infants can also
be a concern given that 25% of infant formulas are soy-based,
and concentrations of the phytoestrogens genistein and daidzein
were nearly 500 times higher in the urine of infants given soybased
formulas compared to infants given formulas derived from
cow’s milk (77). Many animal studies also indicate that
phytoestrogens can compete for ER binding and modulate
normal ER action in target tissues. In transgenic estrogen
reporter mice, genistein treatment inhibited the estrogenic
response elicited by E2 in the liver, indicating that phytoestrogen
exposure can modify the effects of endogenous estrogens (78).
Many phytoestrogens, including flavonoids, display selectivity
for ERβ on the basis of competitive binding assays and
ReView Chem. Res. Toxicol., Vol. 24, No. 1, 2011 11
transcriptional assays (44), although the magnitude of selectivity
in binding assays does not always correspond with selectivity
in transcriptional assays. For example, liquiritigenin (Figure 2,
10) is a flavone found in Glycyrrhizae uralensis that shows a
20-fold selectivity for ERβ in competitive binding assays (RBAs
not determined) (79). Transcriptional assays in 3 different cell
types reveal that although liquiritigenin binds ERR in binding
assays, it induces minimal transcriptional activation of reporter
genes through ERR at concentrations as high as 2.5 µM (79).
Though the crystal structure of the ERβ LBD bound to
liquiritigenin is not yet resolved, it is possible that the ligand
induces conformational changes that selectively recruit coactivators
to ERβ. Indeed, SRC-2 recruitment to ER target genes
selectively occurred in cells expressing ERβ after liquiritigenin
treatment (79), suggesting that ligand binding and transcriptional
potency are not always correlated.
Genistein (Figure 2, 11), an isoflavone found in soy that
shows high binding affinity and selectivity for ERβ (44), has
been shown to have more potent transcriptional activity in cells
transfected with ERβ (80). Crystal structures of genistein
indicate that coactivator binding can stabilize the ligand in the
ligand binding pocket. Genistein is an agonist in transcriptional
assays, but initial crystal structure analysis of genistein and the
ERβ LBD showed a shift in H12 toward the antagonist
conformation in which the coactivator binding site is partially
blocked (28). Crystal structures and computational modeling
of the ERβ LBD with genistein in the presence of a coactivator
LXXLL peptide fragment show H12 in the agonist orientation,
suggesting that the coactivator stabilizes the complex in an active
conformation (81). Genistein also selectively recruits SRC-2 to
ERβ, which may contribute to the transcriptional selectivity for
ERβ (82). Given the importance of coactivators in stimulating
transcription and the potential role of coactivators in stabilizing
ligand-bound ER in an active conformation, the estrogenic
effects of potential EDCs will be highly dependent on the
availability of coactivators which can vary among cell types,
thereby contributing to tissue specific effects.
4. Indirect Actions of EDCs on ER Signaling
4.1. Aryl Hydrocarbon Receptor Ligands and Functional
Cross-Talk with ERs. EDCs can impact ER signaling indirectly
through interactions with the aryl hydrocarbon receptor (AhR),
which is activated by a wide variety of hydrophobic ligands,
most notably polycyclic aromatic hydrocarbons (PAHs) or
halogenated aromatic hydrocarbons (HAHs) (83, 84). AhR
regulates the expression of metabolic enzymes in response to
ligand binding, and the mechanism of AhR signaling shares
many similarities with that of ER signaling (85). The AhR
contains a basic-helix-loop-helix DNA binding domain and
a PER-ARNT-SIM (PAS) homology domain to which ligands
bind (86). AhR is maintained in an inactive cytosolic complex
that includes Hsp90 and p23 in the absence of the ligand
(87-89). Similarly, ERs are maintained in a complex that
includes Hsp90 and p23 in the absence of E2 (90). Upon ligand
binding, AhR undergoes a conformational change that reveals
a nuclear localization sequence, dissociates from the inactive
cytosolic complex, and migrates to the nucleus where it
heterodimerizes with an aryl hydrocarbon receptor nuclear
translocator (ARNT), another bHLH-PAS protein (91). The
heterodimer binds DNA sequences known as dioxin response
elements (DREs) and recruits coactivators, including SRC-1,
SRC-2, and SRC-3 (92), to stimulate the transcription of target
genes. One of the most studied AhR target genes is cytochrome
P450-dependent monooxygenase (CYP) 1A1 (93). The high
affinity AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
is the most potent inducer of Cyp1A1 expression and is a known
EDC (93).
Dioxins and other AhR ligands are primarily antiestrogenic
(94, 95) but can also exhibit weak estrogenic activity (94, 96-98).
AhR and ERs are promiscuous receptors and bind a diverse
array of chemicals, most of which are hydrophobic ligands. The
most notable AhR ligands are environmental chemicals, and
an endogenous ligand has yet to be identified (83). There is
some overlap in the chemicals that can bind AhR and ER. It
has recently been shown that pharmaceutical antiestrogens such
as tamoxifen and many other estrogenic ligands activate AhR
and induce CYP1A1 expression in breast cancer cells that lack
ERR or ERβ expression (99). Dietary flavonoids that are known
estrogenic ligands can also activate AhR such as daidzein and
genistein (44, 100, 101), demonstrating that ER ligands can act
through multiple mechanisms. To illustrate the complexity of
promiscuous ligands, we return to the discussion of WAY-
169916, the nonsteroidal ER dependent selective inhibitor of
NFκB activity discussed in section 2.2. WAY-169916 is
considered a SERM that shows pathway selectivity in terms of
ER mediated signaling (39), but it has also recently been shown
that WAY-169916 can act as a selective AhR modulator
(SAhRM) (102). WAY-169916 can bind AhR with approximately
3 orders of magnitude lower affinity relative to ER and
weakly activates the expression of CYP1A1 in an ER independent
manner. Despite its relatively low activity in terms of
classical DRE driven gene expression, WAY-169916 treatment
has anti-inflammatory effects in a variety of mouse models of
inflammatory diseases and represses the expression of inflammatory
acute phase response genes in an AhR/ARNT dependent
manner (102). WAY-169916 demonstrates that AhR ligands
may selectively modify AhR signaling pathways, thereby acting
as a SAhRM, and ER signaling pathways, thereby acting as a
SERM. Experiments conducted with 3-methylcholanthrene (3MC)
demonstrate the role of cellular context in determining
which pathways are likely to be affected by promiscuous ligands.
3-MC can interact with AhR and ER to stimulate the transcription
of DRE or ERE driven reporter constructs. In HepG2 liver
carcinoma cells, 3-MC acts as an ER agonist and induces the
expression of ERE-luciferase in a dose dependent manner when
cells are transfected with ERR. In contrast, 3-MC cannot
stimulate the expression of ERE-luciferase in HC11 mouse
mammary epithelial cells that express endogenous ERR but
rather inhibits E2-stimulated expression of the ERE reporter
(103). Further analysis of the phenomenon revealed that HepG2
cells treated with 3-MC produced estrogenic metabolites, while
HC11 cells did not. Cell context specific effects have also been
observed for TCDD in MCF7 and HepG2 cells (104).
Though ligand promiscuity strongly influences the cross-talk
between AhR and ER, additional mechanisms may also mediate
such cross-talk (85). First, induction of CYPs may enhance E2
metabolism, thereby reducing the estrogenic response. Hydroxylation
of E2 increases more than 10-fold in MCF7 breast cancer
cells treated with TCDD, possibly due to the induction of
CYP1A1 and CYP1B1 (105, 106). Increased metabolic turnover
of estrogen may not be the primary mechanism of AhR mediated
inhibition of ER signaling given the observation that TCDD
inhibits E2 stimulated cathepsin D expression within 60 min,
preceding the induction of CYP1A1 protein (107). As an
alternative mechanism of AhR-ER cross-talk, AhR may impair
ER mediated transcription through direct binding to ER target
gene promoters (108). DRE sequences that are required for the
inhibitory effects of dioxin on estrogen stimulation of gene
12 Chem. Res. Toxicol., Vol. 24, No. 1, 2011 Shanle and Xu
expression have been identified in the promoter regions of c-fos
(109), pS2 (110), and heat shock protein 27 (111). A third
mechanism of AhR-ER cross-talk may be that activated AhR
competes for available cofactors thereby inhibiting the transcriptional
potential of ER. Activated AhR can recruit coregulators
shared by activated ER, including RIP140 (112), SRC-1,
and SRC-2 (113). It has recently been shown that ARNT can
also act as a coactivator of ERR and ERβ (114) and is a more
potent coactivator for ERβ mediated transcription (115), suggesting
that AhR activation may impair ER mediated transcriptional
activation by sequestering ARNT, and this effect may
be more profound for ERβ signaling. The competition for
cofactors will depend on the amounts and types of cofactors
expressed in the cell, which will be highly dependent on cell
context and tissue type.
4.2. Metabolic Modulators of Estrogen Metabolism. EDCs
can impact ER signaling indirectly through the modification of
E2 metabolism, thereby reducing or enhancing ER signaling in
target tissues (116). Estrogen synthesis is catalyzed by a set of
CYPs and hydroxysteroid dehydrogenases. Cholesterol is the
universal precursor of de noVo synthesis of steroidal hormones
and undergoes a side chain cleavage by CYP11A to form
pregnenolone, which is further modified to form androgens
(117, 118). Androgens are the direct precursors of estrogens,
and the conversion of androgens to estrogens is catalyzed by
aromatase, also known as CYP19A1 (119). Aromatase catalyzes
the formation of two estrogens: estrone and E2. Conversion of
estrone to the more potent E2 is catalyzed by 17β-hydroxysteroid
dehydrogenase (118). In premenopausal women, estrogen
synthesis occurs primarily in the ovaries where it is tightly
regulated by the expression of aromatase, which is up-regulated
by follicle stimulating hormone (FSH) and leutinizing hormone
(LH) during follicle maturation in the preovulatory stage.
Estrogens stimulate the expression of LH receptors, which
promotes a surge of LH and FSH thereby triggering ovulation.
During this critical window of estrogen synthesis, impairment
of aromatase expression or activity could prevent ovulation
(116). Estrogen metabolism occurs primarily in the liver
through oxidation by CYP enzymes, glucuronidation by UDPglucuronosyl
transferases, sulfation by sulfotransferases, or
O-methylation by catechol O-methyltransferase, all of which
generate inactive metabolites that are excreted from the body
(120).
Estrogen synthesis and metabolism may be targets for some
EDCs that inhibit normal estrogen signaling. Atrazine, a triazine
herbicide that is widely present in the environment, is a known
EDC that was shown to induce mammary tumors in F344 female
rats (121) and increase plasma levels of estradiol in female
Sprague-Dawley rats (122). Atrazine and other triazine herbicides
were later shown to induce aromatase activity and gene
expression (123). Pesticides including chlordane or methoxychlor
also induce aromatase in Vitro (124). Enhancing the
activity or expression of aromatase may increase rates of
estrogen synthesis, thereby mediating estrogenic effects. Conversely,
dietary soy supplementation rich in flavonoids decreased
estrogen synthesis in premenopausal women, which may explain
the cancer preventive effects of phytoestrogens (125). Data from
in Vitro experiments suggest that phytoestrogens competitively
bind aromatase preventing normal estrogen synthesis from
androgen substrates. Flavones can bind aromatase with low
micromolar Ki values (126), and, in general, flavones are more
potent inhibitors compared to isoflavones (127). The flavonoids
apigenin, chrysin, and naringenin inhibit aromatase in human
placental microsomes and human breast fibroblasts (128).
Estrogen metabolism and excretion may also be impacted by
EDCs through modified expression of enzymes required for
estrogen clearance in the liver. E2 is initially metabolized by
CYPs including CYP1B1 and CYP1A1, which form 4-hydroxyestradiol
and 2-hydroxyestradiol, respectively (120). In the breast
and uterus, CYP1B1 is highly expressed, and the prominent
metabolite is 4-hydroxyestradiol (129), while 2-hydroxyestradiol
is the most prominent metabolite in the liver, most likely due
to oxidation by a member of the CYP3A family (130). CYP1B1
is induced by activation of AhR and AhR agonists, which can
increase E2 hydroxylation in MCF-7 breast cancer cells (129).
Further metabolism of the hydroxylated estradiol may produce
reactive metabolites that can induce DNA damage (131), which
is thought to be the primary mechanism of ER independent
estrogen mediated carcinogenesis (132).
5. In Vitro and Cell Based Approaches to Identify
EDCs That Impact ER Signaling
Because of concerns associated with EDCs, national and
international programs have developed guidelines for screening
chemicals with endocrine disrupting properties (133, 134). The
guidelines focus on a multitiered approach consisting of Tier
1, in Vitro and in ViVo assays for identifying and classifying
chemicals on the basis of their potential interaction with the
endocrine system; and Tier 2, developing dose-response data
in animal models (134). In 2009, the U.S. Environmental
Protection Agency announced assays for use in Tier 1 screening
for EDCs (135). In Vitro and cell based screens designed to
identify estrogenic compounds include (1) ER binding assays
using rat uterine cytosol preparations, (2) ER transcriptional
assays using hERR-HeLa-9903 cells, which are human cervical
tumor cells with stable expression of ERR and stable integration
of a 5xERE luciferase reporter, (3) steroidogenesis assays with
the adrenocorticoid cell line H295R, and (4) aromatase inhibition
assays using recombinant aromatase (135). Additional in Vitro
and cell based assays are available to identify potential EDCs
including alternative approaches to identify compounds that bind
the receptors and initiate ER mediated transcription as well as
proliferation assays that can sensitively detect estrogenic
chemicals (Table 1). In ViVo assays using rodent models are
Table 1. In Vitro and Cell-Based Assays for Identifying
Estrogenic EDCsa
reference
ER Binding
rat uterine cytosol* 134
fluorescence polarization 138
ER Dimerization
fluorescence resonance energy transfer 139, 140
bioluminescence resonance energy transfer 23, 143
ER Transcriptional Activation
hERR-HeLa-9903 reporter cell line* 134
T47D-KBLuc reporter cell line 144
HELN-ERR and HELN-ERβ reporter cell lines 145, 146
HEK 293 ERR and ERβ ALP reporter cell lines 147
recombinant yeast assays 148
Proliferation Assays
E-SCREEN assay 150, 151
Metabolic Assays
H295R steroidogenesis assay* 134
aromatase competitive inhibition assay* 134
H295R steroidogenic enzyme expression assay 152
JEG-3 steroidogenesis assay 153
a
(*) indicates the assays required by the U.S. Environmental
Protection Agency in Tier 1 screening for potential EDCs (134).
ReView Chem. Res. Toxicol., Vol. 24, No. 1, 2011 13
required to effectively determine if chemicals will act as EDCs
and elucidate tissue specific effects of EDCs. In Vitro assays
cannot replace in ViVo assays due to limitations in considering
the effects of metabolism, clearance, and tissue specific ER
agonist and antagonist activities, but in Vitro and cell based
assays have the advantage of typically being high-throughput,
requiring less time and cost. Additionally, in Vitro assays allow
much more stringent control which allows the determination
of effects on the specific mechanistic processes of ER signaling.
In ViVo identification of EDCs that target ER signaling is
complicated by rat or mouse strain and dietary exposure to
estrogenic compounds, which can have profound effects on
experiments measuring subtle changes in endocrine function
(136). Here, we limit the discussion to the available in Vitro
assays useful for identifying EDCs that target ER signaling in
a relatively high-throughput manner. In ViVo experiments for
identifying EDCs are required for a complete understanding of
the endocrine disrupting properties of chemicals, but in Vitro
prescreening allows for initial identification of chemicals that
target ER signaling and can correlate with in ViVo data for a
large range of chemicals (137). All of the assays discussed herein
are summarized in Table 1.
5.1. Assays to Measure Binding and Dimerization. Binding
assays are useful for identifying EDCs that directly interact with
the LBDs of ERR and ERβ. The U.S. Environmental Protection
Agency Tier 1 assays include an ER binding assay utilizing rat
uterine cytosol, but the assay does not quantitatively measure
ER binding affinity or discriminate between binding to ERR or
ERβ (135). Several approaches are available for sensitively
quantifying receptor binding affinity. Traditional radiolabeled
competitive binding assays have been successfully used to
characterize the ligand binding affinities for ERR and ERβ of
a wide variety of environmental compounds (44). Alternatively,
a nonradioactive fluorescence polarization assay has been
developed that is more amenable to high-throughput screening,
in which changes in polarization of fluorescent E2 are measured
with increasing concentrations of competitor compound (138).
After chemicals bind ERs, dimerization must occur in order
for ERs to mediate transcriptional activation. Multiple assays
to measure dimerization have been developed, which are useful
for high-throughput screening. Fluorescence resonance energy
transfer (FRET) assays can be used to measure protein-protein
interactions and may be used to detect ER homodimer and
heterodimer formation (139) and ER-coactivator interactions
(140) in response to the ligand. FRET utilizes proteins fused to
fluorophores such as green fluorescent protein and measures the
proximity of two proteins; energy transfer from an excited donor
fluorophore on one protein to the nearby acceptor fluorophore
of another protein results in a reduction in the emission of the
donor which can be measured (141). The drawbacks of FRET
assays include autofluorescence, photobleaching, and direct
excitation of the acceptor fluorophore. Bioluminescence resonance
energy transfer (BRET) is an alternative assay to measure
dimerization that overcomes some of the limitations associated
with FRET. BRET assays utilize an enzyme catalyzed bioluminescent
donor such as luciferase that emits photons only in
the presence of substrate. When the substrate is added, luciferase
catalyzes a reaction that emits light which can excite an acceptor,
such as yellow fluorescent protein, if it is in close proximity to
luciferase. Proteins of interest are fused to luciferase or the
acceptor fluorophore, respectively (142). BRET assays have
been successfully used to measure ER homodimerization and
heterodimerization in live cells, and high-throughput screening
for chemicals that induce ER dimers is possible using this highly
optimized assay (23, 143).
5.2. Transcriptional Assays. Transcriptional assays in cell
lines using reporter genes such as luciferase or β-galactosidase
downstream of an ERE provide a tool to identify compounds
that act as ER agonists or antagonists. Transcription of target
genes is dependent on a variety of factors, including promoter
context, cell context, and the availability of cofactors. Several
cell lines have been generated to identify chemicals with
estrogenic activity and quantify the relative potency of such
compounds. T47D breast cancer cells express both ERR and
ERβ, and chemicals that activate either receptor can be identified
using T47D-KBLuc cells in which a luciferase reporter downstream
of three tandem EREs is stably integrated (144). T47DKBLuc
cells were shown to be highly sensitive and stable over
many passages, making them amenable for use in highthroughput
screening (144). Currently, Tier 1 transcriptional
assays aimed at detecting chemicals with estrogenic activity are
limited to HeLa cells that express ERR alone, neglecting the
potential for EDCs to selectively activate ERβ. In order to
identify compounds that display selectivity for ERR or ERβ,
HeLa cells with stable ERE-luciferase (HELN) were created
with stable expression of ERR or ERβ (HELN-ERR and HELNERβ,
respectively); in this system, genistein was shown to have
high selectivity for ERβ (145). HELN-ERR and HELN-ERβ
cells have also been used to demonstrate the antiestrogenic
activities of persistent pesticides on ERβ mediated transcription
(146). A similar approach has been applied to human embryonic
kidney 293 cells by stable expression of ERR or ERβ and stable
integration of a human placental alkaline phosphatase reporter
downstream of a single ERE (147). Alternative reporter systems
include recombinant yeast assays in which a β-galactosidase
reporter is expressed after ligand dependent activation of ER
(148), but mammalian systems provide a better model given
the role of coactivators and interactions of signaling pathways
in mediating the transcriptional activation of ERs.
5.3. Proliferation Assays. The E-SCREEN is a highly
sensitive cell based assay for detecting estrogenic compounds
and has been utilized for detecting estrogenic activity in
wastewater and environmental samples (149, 150). MCF7 breast
cancer cells proliferate rapidly after exposure to estrogenic
chemicals. The E-SCREEN assay measures the proliferation of
MCF7 cells after treatment with a range of concentrations of
the chemical or sample of interest, using E2 as a positive control.
Using this assay, a variety of compounds were found to be
estrogenic including alkylphenols, phthalates, some PCB congeners,
and hydroxylated PCBs. These compounds were further
shown to bind ER and activate transcription of pS2, an estrogen
responsive gene, in MCF7 cells (151). Proliferation assays such
as the E-SCREEN can be highly sensitive, and results may be
relevant given that the system utilizes cells of human origin
which express endogenous coactivators that may mediate
estrogenic potency.
5.4. Metabolic Assays. As mentioned previously, Tier 1
assays for identifying EDCs include two in Vitro assays to detect
metabolic inhibitors. A competitive binding assay aims to detect
chemicals that will directly interfere with the conversion of
androgens to estrogens by competing for the catalytic activity
of aromatase, and a steroidogenesis assay utilizing the adrenocarcinoma
cell line H295R aims to detect chemicals that can
interfere with estrogen or androgen production by measuring
hormone production after exposure to suspected EDCs (135).
H295R cells provide a tool to measure both steroid production
and expression levels of steroidogenesis enzymes. Quantitative
14 Chem. Res. Toxicol., Vol. 24, No. 1, 2011 Shanle and Xu
RT-PCR may be used to measure changes in the expression of
enzymes required for normal estrogen synthesis (152). Human
placental JEG-3 cells also express relatively high levels of
aromatase and may be useful for identifying inhibitors of
estrogen synthesis, but the cells are vulnerable to the toxicity
of compounds making high-throughput screening more difficult
(153).
6. Conclusions and Future Perspectives
The impacts of EDCs on ER signaling are complex and often
occur through multiple direct and indirect mechanisms, making
it difficult to predict the end points of EDC toxicity in cultured
cells and animals. Though ERs are relatively promiscuous
receptors, insight from crystal structures is useful for defining
characteristic features of estrogenic ligands that act through
direct interaction with ER LBDs. Such chemicals may be
detected through screening assays aimed at measuring ER
binding and dimerization such as fluorescence polarization and
FRET/BRET. Current guidelines for detecting chemicals that
interact with ER through binding do not discriminate between
receptor subtypes despite the fact that many EDCs such as
phytoestrogens display selectivity for ERβ. Tier 1 assays
required by the U.S. Environmental Protection Agency also
neglect transcriptional activation of ERβ. EDCs may also act
indirectly through other signaling pathways such as AhR or
through modification of normal estrogen synthesis. Cross-talk
between AhR and ER is highly complex and dependent on cell
context, availability of cofactors, and promoter context, which
makes it difficult to develop universal, effective in Vitro assays
for identifying EDCs that impact ER signaling through the
stimulation of AhR; such indirect EDCs may be detected more
effectively using in ViVo models. Finally, EDCs that impact ER
signaling indirectly through modification of estrogen synthesis
may be identified using assays currently required by the U.S.
Environmental Protection Agency’s Tier 1 protocols.
Despite multiple approaches to identify EDCs using in Vitro
and cell based assays, it is difficult to predict the toxic end points
induced by EDCs in ViVo. Though a chemical may not display
estrogenic effects at low concentrations in highly controlled
experiments, estrogenic effects may be elicited in the background
of broad chemical exposure that occurs in our environment.
EDCs may have additive or synergistic effects when
combined (see ref 46), and such complicated effects are
compounded by individual differences in uptake, metabolism,
and excretion. Tissue specific effects of EDCs are also difficult
to predict using in Vitro and cell based assays, which underscores
the importance of examining the effects of EDCs in ViVo with
animal models. Though in Vitro and cell based assays cannot
replace in ViVo models, approaches to predict tissue specific
effects using in Vitro techniques are being developed. A recent
study utilized hydrogen/deuterium exchange (HDX) mass
spectrometry to predict the tissue selectivity of pharmaceutical
SERMs used for the treatment of breast cancer (154). HDX mass
spectrometry measures the rate of hydrogen and deuterium
exchange at amide groups, and variations in the exchange
kinetics are a function of hydrogen bonding. Exchange rates
are higher in regions where the ligand is bound to the receptor
and in regions in which the receptor undergoes conformational
changes. On the basis of the exchange kinetics, ligands could
be classified on the basis of tissue selectivity in most cases (154).
Such an approach provides insight into the dynamic structural
effects of ligands interacting with ERs and may prove useful
for predicting the tissue specificity of EDCs. In Vitro and cell
based assays measuring specific mechanisms of ER signaling
such as ligand binding, dimerization, DNA binding, and
transcriptional activation are important initial screens for
identifying EDCs. As our understanding of estrogen signaling
mechanisms expands and technology advances, in Vitro and cell
based assays will become more powerful tools for early
identification of EDCs.
Acknowledgment. This publication was made possible by
grant number T32 ES007015 from the National Institute of
Environmental Health Sciences (NIEHS), NIH. Its contents are
solely the responsibility of the authors and do not necessarily
represent the official views of the NIEHS, NIH. W.X. is
supported by NIH grants R01CA125387 and R03MH089442,
Markos Family Breast Cancer Research Grant, and Shaw
Scientist Award from Greater Milwaukee Foundation.
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