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Center for Biotechnology and Department of Medical Nutrition (G.G.J.M.K., J.-Å.G.), Karolinska Institute and KaroBio AB (B.C.) Huddinge, Sweden; Hubrecht Laboratory, Netherlands Institute for Developmental Biology (B.v.d.B., P.T.v.d.S., J.G.L.) Utrecht, The Netherlands; Chemical Industry Institute of Toxicology (J.C.C.), Research Triangle Park, North Carolina; Department of Veterinary Physiology and Pharmacology (S.H.S.), Texas A&M University, College Station, Texas 77843-4466
Address all correspondence and requests for reprints to: Dr. George Kuiper, Center for Biotechnology, NOVUM, S-14186 Huddinge, Sweden. E-mail: george.kuiper{at}csb.ki.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß, which differ in the C-terminal ligand-binding domain
and in the N-terminal transactivation domain. In this study, we
investigated the estrogenic activity of environmental chemicals and
phytoestrogens in competition binding assays with ER or ERß
protein, and in a transient gene expression assay using cells in which
an acute estrogenic response is created by cotransfecting cultures with
recombinant human ER or ERß complementary DNA (cDNA) in the
presence of an estrogen-dependent reporter plasmid.
Saturation ligand-binding analysis of human ER and ERß protein
revealed a single binding component for
[3H]-17ß-estradiol (E2) with high affinity
[dissociation constant (Kd) = 0.05 - 0.1 nM].
All environmental estrogenic chemicals [polychlorinated
hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and
derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone]
compete with E2 for binding to both ER subtypes with a
similar preference and degree. In most instances the relative binding
affinities (RBA) are at least 1000-fold lower than that of
E2. Some phytoestrogens such as coumestrol, genistein,
apigenin, naringenin, and kaempferol compete stronger with
E2 for binding to ERß than to ER. Estrogenic
chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and
2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity
of ER and ERß at concentrations of 100-1000 nM.
Phytoestrogens, including genistein, coumestrol and zearalenone
stimulate the transcriptional activity of both ER subtypes at
concentrations of 1–10 nM. The ranking of the estrogenic
potency of phytoestrogens for both ER subtypes in the transactivation
assay is different; that is, E2 >> zearalenone =
coumestrol > genistein > daidzein > apigenin =
phloretin > biochanin A = kaempferol = naringenin
> formononetin = ipriflavone = quercetin = chrysin for
ER and E2 >> genistein = coumestrol >
zearalenone > daidzein > biochanin A = apigenin =
kaempferol = naringenin > phloretin = quercetin =
ipriflavone = formononetin = chrysin for ERß.
Antiestrogenic activity of the phytoestrogens could not be detected,
except for zearalenone which is a full agonist for ER and a mixed
agonist-antagonist for ERß. In summary, while the estrogenic potency
of industrial-derived estrogenic chemicals is very limited, the
estrogenic potency of phytoestrogens is significant, especially for
ERß, and they may trigger many of the biological responses that are
evoked by the physiological estrogens.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Rat ERß cDNA
encodes a protein of 485 amino acid residues with a calculated
molecular weight of 54200. Rat ERß protein is highly homologous to
rat ER protein, particularly in the DNA binding domain (95% amino
acid identity) and in the C-terminal ligand binding domain (55%
homology). In addition, recently a variant rat ERß cDNA was cloned
that has an in-frame insertion of 54 nucleotides that results in the
predicted insertion of 18 amino acids within the ligand-binding domain
(9, 10). Mouse (11, 12) and human homologs (13, 14) of rat ERß have
been cloned, and similar homologies in the various domains of the
subtypes were found. Expression of ERß was investigated by Northern
blotting, RT-PCR, and in situ hybridization; prominent
expression was found in prostate, ovary, epididymis, testis, bladder,
uterus, lung, thymus, colon, small intestine, vessel wall, pituitary,
hypothalamus, cerebellum, and brain cortex (4, 10, 11, 12, 13, 14, 15, 16). Saturation
ligand binding experiments revealed high affinity and specific binding
of 17ß-estradiol (E2) by ERß protein, and ERß is able
to stimulate transcription of an estrogen response element containing
reporter gene in an E2-dependent manner (10, 11, 12, 13, 15). More
extensive studies showed that some synthetic estrogens and naturally
occurring steroidal ligands have different relative affinities for
ER vs. ERß, although most ligands (including various
antiestrogens) bind with very similar affinity to both ER subtypes
(15).
There is increasing concern over the putative effects of various
chemicals released into the environment on the reproduction of humans
and other species. Threats to the reproductive capabilities of birds,
fish, and reptiles have become evident and similar effects in humans
have been proposed (17, 18, 19, 20, 21). In the past 50 yr, the incidence of
testicular cancer and developmental male reproductive tract
abnormalities appear to have increased in some developed countries
(19). Several reports have also provided evidence for a decline in
semen quality and/or sperm count over the same period, although this
change may not be universal (19 and references therein). Male offspring
born to mothers who were given diethylstilbestrol (DES), a very potent
synthetic estrogen, to prevent miscarriages have an increased incidence
of undescended testes, urogenital tract abnormalities, and reduced
semen quality compared with those from mothers who did not take DES (22
and references therein). In mice injected with DES between days 9 and
16 of gestation, there is an increased risk of intraabdominal testes,
sterility, and abnormalities in the urogenital tract of the offspring
(22 and references therein). The similarities between the observations
made in DES offspring and the abnormalities being observed in the
general population have led to the hypothesis that one potential cause
of the rise in male reproductive tract abnormalities might be
inappropriate exposure to estrogens or suspected environmental
estrogenic chemicals (from pesticides, components of plastics, hand
creams, etc.) especially during fetal and/or neonatal life (17, 18, 19, 20, 21).
Examples of suspected environmental estrogenic chemicals include
OH-PCBs (polychlorinated hydroxybiphenyls), DDT and derivatives,
certain insecticides and herbicides as Kepone and methoxychlor, certain
plastic components as bisphenol A, and some components of detergents
and their biodegradation products as, for instance, alkylphenols
(17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29). All these compounds bind weakly to the ER protein
extracted from rat uterus or human breast tumor cells or with
recombinant ER protein (23, 24, 25, 26, 27, 28, 29). No data are yet available on the
potential interaction of estrogenic chemicals with ERß, and
interactions of xenoestrogens with this subtype may be related to some
recent observations. In the rat and mouse prostate, ERß messenger RNA
(mRNA) is highly expressed in the secretory epithelial cells (8, 30),
and it has been shown that fetal or neonatal exposure to
E2/DES or estrogenic chemicals causes not only permanent
changes in the size of the prostate but also in the expression level of
certain genes (30, 31, 32). In the fetal rat testis, ERß is expressed in
Sertoli cells and gonocytes (33), and maternal exposure to DES or
4-octylphenol alters the expression of steroidogenic factor I (SF-1) in
Sertoli cells of the fetal rat testis (34). In the human
mid-gestational fetus, high amounts of ERß mRNA are present in
the testes, but the cellular localization is unknown (35).
Human diet contains several plant-derived, nonsteroidal weakly
estrogenic compounds (1). They are either produced by plants themselves
(phytoestrogens), or by fungi that infect plants (mycoestrogens).
Chemically, the phytoestrogens can be divided into three main classes:
flavonoids (flavones, isoflavones, flavanones and chalcones) such as
genistein, naringenin, and kaempferol; coumestans (such as coumestrol);
and lignans (such as enterodiol and enterolactone). Mycoestrogens are
mainly zearalenone (resorcylic acid lactone) or derivatives thereof,
which have been associated with estrogenizing syndromes in cattle fed
with mold-infected grain (1). Phytoestrogens and mycoestrogens act as
weak mitogens for breast tumor cells in vitro, compete with
17ß-estradiol for binding to ER protein, and induce activity of
estrogen-responsive reporter gene constructs in the presence of ER
protein (36, 37, 38). Intake of phytoestrogens is significantly higher in
countries where the incidence of breast and prostate cancers is low,
suggesting that they may act as chemopreventive agents (39). The
chemopreventive effect of dietary soy, which is rich in phytoestrogens,
has been demonstrated on the development of chemically or
irradiation-induced mammary tumors in mice (39 and references therein),
and as a delayed development of dysplastic changes in the prostate of
neonatally estrogenized mice (40). The expression of ERß in rat,
mouse, and human prostate might be of importance in this regard.
Phytoestrogens are believed to exert their chemopreventive action by
interacting with estrogen receptors, although alternative mechanisms,
most notably inhibition of protein tyrosine kinase activity, have been
proposed (39, 41).
In the present study, we have evaluated the estrogenic activity of
suspected environmental estrogens and phytoestrogens in competition
binding assays with ER or ERß protein, and in a transient gene
expression assay using cells in which an acute estrogenic response is
created by cotransfecting cultures with recombinant human ER or
ERß cDNA in the presence of an estrogen-dependent reporter
plasmid.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-estradiol (1, 3,
5(10)-estratriene-3,17-diol), 16-keto-17ß-estradiol (1, 3,
5(10)-estratriene-3,17ß-diol-16-one), 17-epiestriol (1, 3,
5(10)-estratriene-3,16,17-triol), 16-bromoestradiol (1, 3,
5(10)-estratriene-16-bromo-3,17ß-diol), 2-OH-estrone (1, 3,
5(10)-estratriene-2,3-diol-17-one), progesterone, 5-androstenediol
(5-androstene-3ß, 17ß-diol) and testosterone were obtained from
Steraloids Inc. (Wilton, NH).
The synthetic estrogen diethylstilbestrol (4, 4'-(1, 2-diethyl-1,
2-ethene-diyl)-bisphenol) was obtained from Steraloids. The
antiestrogens tamoxifen
(1-p-ß-dimethylamino-ethoxy-phenyl-trans
-1,2-diphenyl-1-butene), 4-OH-tamoxifen
(1-(p-dimethylaminoethoxy-phenyl)1-(4-hydroxyphenyl)-2-phenyl-1-butene),
raloxifene
(6-hydroxy-3-[4-[2-(1-piperidinyl)ethoxy]phenoxy]-2-(4-hydroxy
phenyl)-benzothiophene) and ICI-164384 (N-n- butyl-11-(3,
17ß-dihydroxyestra-1, 3,
5(10)trien-7-yl)-N-methyl-undecanamide) were obtained
from Sigma Chemical Co. (St. Louis, MO) or synthesized by KaroBio AB.
The steroidal antiestrogen ICI-182780 was kindly supplied by Zeneca
Pharmaceuticals (Cheshire, UK).
The flavonoids genistein (4', 5, 7-trihydroxyisoflavone), daidzein (4', 7-dihydroxyisoflavone), formononetin (7-hydroxy-4'-methoxyisoflavone), biochanin A (5, 7-dihydroxy-4'-methoxyisoflavone), apigenin (4', 5, 7-tri-hydroxyflavone), chrysin (5, 7-dihydroxyflavone), kaempferol (3, 4', 5, 7-tetrahydroxyflavone), quercetin (3, 3', 4', 5, 7-pentahydroxyflavone), naringenin (4', 5, 7-trihydroxyflavanone), phloretin (2', 4, 6'-trihydroxy-3-(p-hydroxyphenyl)-propiophenone), ipriflavone (7-isopropoxyisoflavone), and the nonhydroxylated compound flavone (2-phenyl-1, 4-benzopyrone) were obtained from Sigma or Roth Chemicalien (Karlsruhe, Germany). The phytoestrogen coumestrol (2-(2, 4-dihydroxyphenyl)-6-hydroxy-3-benzofurancarboxylic acid lactone) was obtained from Eastman Kodak (Rochester, NY) and zearalenone (6-[10-hydroxy-6-oxo-trans-1-undecenyl)-2,4-dihydroxybenzoic acid lactone) from Sigma.
The insecticide DDT and metabolites 2,4'-DDT/o, p'-DDT (1-chloro-2-(2,
2, 2-trichloro-1-(4-chlorophenyl)ethyl)benzene), 4,4'-DDT/p,
p'-DDT (1, 1'-(2, 2,
2-tri-chloroethylidene)bis(4-chlorobenzene)), 2,4'-DDE/o, p'-DDE
(2(2-chloro-phenyl)-2-(4-chlorophenyl)-1,1-dichloro-ethylene),
4,4'-DDE/p, p'-DDE (1,
1'-(dichloroethenylidene)-bis(4-chlorobenzene)), 2,4'-TDE/o, p'-TDE
(1-chloro-2-(2, 2-dichloro-1-(4-chlorophenyl)ethyl)-benzene),
4,4'-TDE/p, p'-TDE (1, 1'-(2,
2-dichloroctylidene)-bis(4-chlorobenzene)), chlordecone (Kepone)
(decachloro-octahydro-1,3,4-metheno-2H-cyclobuta(cd)pentalene),
endosulfan (1, 4, 5, 6, 7, 7-hexachloro-5-norbornene-2, 3-dimethanol
cyclic sulfite) and methoxychlor (1, 1, 1-trichloro-2,
2-bis(p-methoxyphenyl)ethane) were obtained from CIIT (Chemical
Industry Institute of Toxicology, Research Triangle Park, NC). The
plastic component bisphenol A (2, 2-bis(4-hydroxy-phenyl)propane) and
the alkylphenolic compounds 4-tert-octylphenol,
4-octylphenol, 4-tert-amyl-phenol, 4-tert-
butylphenol and nonylphenol were obtained from Aldrich (Tyres
,
Sweden).
The hydroxylated polychlorinated biphenyl (OH-PCB) congeners OH-PCB-A (2, 2', 3', 4', 5'-pentachloro-4-biphenylol), OH-PCB-B (2, 2', 3', 4', 6'-pentachloro-4-biphenylol), OH-PCB-C (2, 2', 3', 5', 6'-pentachloro-4-biphenylol), OH-PCB-D (2, 2', 4', 6'-tetrachloro-4-biphenylol), OH-PCB-E (2', 3, 3', 4', 5'-pentachloro-4-biphenylol), OH-PCB-F (2', 3, 3', 4', 6'-pentachloro-4-biphenylol), OH-PCB-G (2', 3, 3', 5', 6'-pentachloro-4-biphenylol), OH-PCB-H (2', 3, 4', 6'-tetrachloro-4-biphenylol), OH-PCB-K (2', 4', 6'-trichloro-4-biphenylol), OH-PCB-L (2', 3', 4', 5'-tetrachloro-4-biphenylol), OH-PCB1 (2, 3, 3', 4', 5-pentachloro-4-biphenylol), OH-PCB2 (2, 2', 3, 4', 5, 5'-hexachloro-4-biphenylol), OH-PCB3 (2, 2', 3', 4, 4', 5, 5'-heptachloro-3-biphenylol), OH-PCB4 (2', 3, 3', 4', 5-pentachloro-4-biphenylol), OH-PCB5 (2, 2', 3, 3', 4', 5-hexachloro-4-biphenylol), OH-PCB6 (2, 2', 3, 3', 4', 5, 5'-heptachloro-4-biphenylol) and OH-PCB7 (2, 2', 3, 4', 5, 5', 6-heptachloro-4-biphe-nylol) were synthesized via Cadogan coupling as described (42, 43). The purity was greater than 98% as determined by gas-liquid chromatography. The nonchlorinated compounds 4,4'-biphenol and 4-biphenylol were obtained from Aldrich. The structural formula and chemical properties of the compounds used can be found in the Merck Index or elsewhere (1, 37, 41, 42, 43).
Expression and generation of ER and ERß protein extracts
A 1.5-kb DNA fragment encoding the human homolog of rat ERß
protein (485 amino acid residues) was excised with
SacII/SpeI from pGEM-T/hERß (14) and isolated
from agarose gel. The fragment was ligated to a
BamHI/SacII adapter, recut with
BamHI/SpeI and ligated into the
BamHI/XbaI sites of the baculovirus donor vector
pFastBac 1 (Life Technologies, Gaithersburg, MD). Recombinant
baculovirus was generated using the BAC-TO-BAC expression system (Life
Technologies) in accordance with manufacturer’s instructions.
The human ER coding sequence was derived from the mammalian
expression vector pMT-hER. The plasmid was linearized with
SacI, and a BamHI linker was ligated after T4
DNA-polymerase treatment. The 1.9-kb fragment encoding hER was
excised with BamHI and cloned into the baculovirus transfer
vector pVL941 (kindly provided by Dr. M. D. Summers, Texas A&M
University, College Station, TX). The recombinant transfer vector
pVL941/hER was cotransfected together with wild-type AcNPV
DNA into Sf9 cells and polyhedrin negative plaques were isolated after
several rounds of plaque purification. The recombinant baculoviruses
were amplified and used to infect Sf9 cells. Infected cells were
harvested 48 h post infection. A nuclear fraction was obtained as
described (44), the resulting nuclei were extracted with buffer (17
mM K2HPO4, 3 mM
KH2PO4, 1 mM MgCl2, 0.5
mM EDTA, 6 mM monothioglycerol, 400
mM KCl, 8.7% glycerol; pH = 7.6) and the
concentration of ER protein in the extract was measured as specific
3H-17ß-estradiol binding with the solubilized receptor
based assay (see below). The ER extract contained 400 pmol
receptor/ml and the ERß extract contained 800 pmol receptor/ml. The
extracts were aliquoted and stored at -80 C.
Nonseparation solid-phase ligand binding competition
experiments
These experiments were performed as described (45). In brief,
the nuclear extracts were diluted (ER extract 50-fold and ERß
extract 90-fold) in coating buffer (17 mM
K2HPO4, 3 mM
KH2PO4, 40 mM KCl, 6 mM
monothioglycerol, pH 7.6). The diluted extracts (200 µl/well) were
added to Scintistrip wells (Wallac Oy, Turku, Finland) and incubated
for 18 h at ambient temperature.
Following noncovalent adhesion of receptor proteins the wells were washed twice with buffer A (17 mM K2HPO4, 3 mM KH2PO4, 140 mM KCl, 6 mM monothioglycerol, pH 7.6). Serial dilutions of the compounds to be tested were made in DMSO to concentrations 50-fold higher than the desired final concentrations. The DMSO solutions were diluted 50-fold in buffer A containing 3 nM 3H-17ß-estradiol [NEN-Life Science Products, Boston, MA; specific activity (S.A.) = 85 Ci/mmol]. The binding experiments were initiated by adding the incubation mixtures (175 µl) to the washed wells. Incubation was for 18 h at ambient temperature. The Scintistrip plates were counted in a MicroBeta counter fitted with six detectors (Wallac Oy, Turku, Finland). The data were evaluated by a nonlinear four-parameter logistic model (46) to estimate the IC50 value (the concentration of competitor at half-maximal specific binding). Relative binding affinity (RBA) of each competitor was calculated as the ratio of concentrations of E2 and competitor required to reduce the specific radioligand binding by 50%, and the RBA value for E2 was arbitrarily set at 100.
Ligand binding experiments with solubilized receptor using gel
filtration for separation of bound and free radioligand
These experiments were performed, with minor modifications, as
described previously (47). In brief: insect cell extracts were diluted
in buffer B (20 mM HEPES, pH 7.5; 150 mM KCl, 1
mM EDTA, 6 mM monothioglycerol, 8.7%
[vol/vol) glycerol) to a final ER concentration of 0.3–0.4
nM. Serial dilutions of the compounds to be tested were
made in DMSO to concentrations 50-fold higher than the desired final
concentrations. The DMSO solutions were diluted 50-fold with buffer B
and 3H-17ß-estradiol (NEN-Life Science Products; S.A. =
85 Ci/mmol) was added to a final concentration of 3 nM.
Unprogrammed rabbit reticulocyte lysate (Promega, Madison, WI; 1
µl/200 µl) was added to increase the protein concentration.
Incubation was for 18–20 h at 6 C. Bound and free radioligand were
separated on Sephadex G-25 columns as described (46), and the
radioactivity in the eluate was measured after addition of 4 ml Wallac
Supermix scintillation cocktail in a Wallac Rackbeta 1217 counter
(Wallac Oy, Turku, Finland). The IC50 and RBA values were
calculated as described above.
For saturation ligand binding analysis, the insect cell extracts were diluted to a final ER concentration of about 0.1 nM, and incubated for 18 h at 4 C with a range of 3H-17ß-estradiol (S.A. = 130 Ci/mmol) concentrations in the presence or absence of a 300-fold excess of unlabeled E2. The dissociation constant (Kd) was calculated as the free concentration of radioligand at half-maximal specific binding by fitting data to the Hill equation (48) and by linear Scatchard transformation (49).
Transient gene expression assay in 293 human embryonal kidney
cells
The estrogen-responsive reporter gene construct
(3xERE-TATA-LUC) which contains three copies of a consensus estrogen
response element (ERE) containing oligonucleotide and a TATA box in
front of the luciferase cDNA, is described in more detail elsewhere
(van der Burg et al., in preparation). The human ERß
expression plasmid pSG5-hERß contains a 1.5 kb human ERß cDNA,
encoding the 485 amino acid residue human ERß protein as described
(14). The human ER expression plasmid pSG5-HEGO (kindly provided by
Dr. P. Chambon, IGBMC, Strasbourg, France) was used. Human 293
embryonal kidney cells were obtained from the ATCC (American Type
Culture Collection, Rockville, MD), and cultured in a 1:1 mixture of
DMEM and Ham’s F12 medium (DF) supple-mented with 7.5% FCS. The cells
were trypsinized and suspended in phenol red free DF medium containing
30 nM selenite, 10 µg/ml transferin and 0.2% BSA,
supplemented with 5% charcoal stripped FCS. They were plated in 24
well tissue culture plates and 24 h later the cultures were
transfected by the calcium phosphate precipitation method (50) with 1
µg 3xERE-TATA-LUC, 0.2 µg SV2-LacZ (51) internal control plasmid
and 0.1 µg of the respective ER expression plasmid. After 16 h
the medium was changed and the compounds to be tested (dissolved in
ethanol) were added directly to the medium at a 1:1000 dilution. After
24 h, the cells were scraped in lysis solution (1% (vol/vol)
Triton X-100, 25 mM glycylglycine, 15 mM
MgSO4, 4 mM EGTA and 1 mM DTT). The
luciferase activity of the cell lysates was measured with the Luclite
luciferase reporter gene assay system (Packard Instruments, Meriden,
CT) according to manufacturer’s instructions, and the
ß-galactosidase activity was measured to correct for variations in
transfection efficiencies (51).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein, have been
expressed in large quantities in the baculovirus-Sf9 insect cell system
and reported to be biologically active and structurally
indistinguishable from the authentic receptor proteins (52).
Furthermore, it has been demonstrated that posttranslational processing
of proteins produced in Sf9 insect cells closely parallels these events
in mammalian cells (53). It was therefore decided to use human ER
and ERß protein expressed in insect cells for the ligand binding
experiments.
In Fig. 1
, the result of a saturation
ligand binding experiment with [3H]-17ß-estradiol in
the solubilized receptor ligand-binding system (see Materials
and Methods) is shown. At the receptor concentrations employed
(0.05–0.1 nM) the Kd values calculated from
the saturation curves were 0.05 nM for ER and 0.07
nM for ERß protein. Linear transformation of saturation
data (Scatchard plots in Fig. 1) revealed a single population of
binding sites for 17ß-estradiol with a Kd of 0.05
nM for the ER protein and 0.09 nM for ERß
protein. In a previous report (15) we found a 4-fold higher affinity
for ER compared with ERß, however, in that study
16-[125I]-iodo-17ß-estradiol was used as ligand
instead of [3H]-17ß-estradiol.
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and ERß protein and ERß
proteins in insect cell extracts are attached to the wells of
scintillating microtitration plates. The signal detection is based on
the fact that 3H emits low energy electrons that have a
very short range in solution and therefore only radioligand bound to
receptors triggers a scintillation process.
Overall ER and ERß show the relative binding affinities (Table 1) for the steroidal ligands and
antiestrogens characteristic for an ER protein (1, 5, 15). The
estradiol binding is stereospecific and the most potent synthetic
estrogen DES binds with equal relative affinity to both ER proteins.
The measured 7-fold greater affinity of 16-bromo-17ß-estradiol for
ER is in line with the measured 4-fold higher Kd (=
lower affinity) of ERß compared with ER for the radioligand
16-iodo-17ß-estradiol (15). The selective estrogen receptor
modulator (SERM) raloxifene and various E2 metabolites
(17-epiestriol and 16-keto-17ß-estradiol) that have been shown to
stimulate ER mediated TGF-ß3 gene transcription in bone cells via
a novel non-ERE-dependent pathway (54), also interact with the ERß
protein.
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and
ERß protein (25, 55) and induces estrogenic effects in female rats. The binding affinity
of o, p'-DDT to both ER subtype is 5000- to 10,000-fold lower in
comparison to E2, whereas for the other DDT isomers and
metabolites significant radioligand competition was not detected at
concentrations up to 10 µM. Apart from DDT, other
organochlorine insecticides exhibit estrogenic activity, most notably
chlordecone (19, 26). Of these (methoxychlor, chlordecone, and
endosulfan) only chlordecone bound to both ER subtypes (Table 1
).
Polychlorinated biphenyls (PCBs) are highly toxic halogenated aromatic
compounds that are widely distributed in the global ecosystem.
Metabolism of PCBs by humans and rodents results in formation of
hydroxylated PCBs (OH-PCBs), and several OH-PCBs elicit estrogenic
responses in the rat uterus (23). We have investigated the ER binding
affinity of a series of OH-PCBs including those identified in human
serum (24, 42, 43). In general only minimal, if any competition, was
detected (Table 1), except for OH-PCB-K (2', 4',
6'-trichloro-4-biphenylol) and OH-PCB-L (2', 3', 4',
5'-tetrachloro-4-biphenylol), which bound to ER and ERß proteins
with affinities only 20- to 40-fold lower than E2. The
OH-PCBs K and L have chlorine atom substitutions only in the
nonphenolic ring, while all other OH-PCBs tested have chlorine
substitutions in both the phenolic and nonphenolic rings. Substitution
of one chlorine atom at the para or meta position in the phenolic ring
of OH-PCB-K and OH-PCB-L, respectively, lowers the binding affinity
about 20-fold for both ER subtypes (compare OH-PCB-K with OH-PCB-D and
OH-PCB-L with OH-PCB-E in Table 1). The very low binding affinity for
ER as well as ERß protein of the OH-PCBs tested, except for those
which have no chlorine atom substitutions in the phenolic ring, is in
agreement with previous studies in which radioligand competition
experiments were performed using rat or mouse uterus cytosol as a
source of ER protein (23, 24, 43).
Alkylphenols are composed of an alkyl group that can vary in size,
branching, and position joined to a phenolic ring. Nonylphenol and
octylphenol are estrogenic in the breast cancer cell proliferation
assay (17, 21, 29), in a recombinant yeast screen with human ER (27)
and in the rat uterus growth bioassay (56), although they are 1000- to
10,000-fold less potent than E2. Alkylphenols compete with
E2 for binding to both ER subtypes to the same extent; that
is nonylphenol > 4-octylphenol >
4-tert-octylphenol > 4-tert-amylphenol
= 4-tert-butylphenol (Table 1). The binding affinity
increases with the number of C-atoms in the alkylgroup, although it is
maximally 1000- to 2000-fold lower for both ER subtypes as compared
with E2. The affinity for ERß seems to be higher, but
more alkylphenols should be tested to see if this is a general
finding.
Bisphenol A is the monomer used in the production of polycarbonate
plastics, and it shows estrogenic activity in MCF-7 human breast cancer
cells as well as in rats (28, 57). Bisphenol A has an affinity
10,000-fold lower than that of E2 for both ER subtypes
(Table 1) and 4,4'-biphenol, which lacks the propane group between the
phenolic rings, has a similarly low affinity for ER and ERß.
Differential binding of several phytoestrogens to ER and ERß
protein
The binding affinity of coumestrol to ERß is 7-fold higher in
comparison to ER, whereas for zearalenone only a very small
difference in affinity is detectable (Table 2). Several flavonoids, especially
genistein, apigenin and kaempferol have a higher binding affinity (20-
to 30-fold more) for ERß in the solid-phase binding assay (Table 2).
The exact position and number of the hydroxyl substituents on the
flavone or isoflavone molecule seem to determine the ER binding
affinity. For example, the isoflavone genistein has a particular high
binding affinity for ERß, but elimination of one hydroxyl group
(daidzein, biochanin A) or two hydroxyl groups (formononetin) causes a
great loss in binding affinity. The flavone apigenin has moderate
affinity for both ER subtypes and addition of hydroxyl groups
(kaempferol, quercetin) does not increase but decreases the binding
affinities.
|
),
which was intended to be an initial screening assay, several compounds
were also analyzed in more traditional solubilized receptor ligand
binding assays (Fig. 2). This is
essentially the same assay as the saturation ligand-binding experiments
described in Fig. 1, but now in the competition mode. Again, the
binding affinity of 16-bromo-17ß-estradiol was significantly
higher (about 4-fold) for ER, whereas the binding affinity of
5-androstenediol is significantly higher for ERß, as previously
described (15). Furthermore, the relative binding affinity of
raloxifene for both ER subtypes is similar in both binding assays. For
the phytoestrogens the differences in relative binding affinities (RBA)
between the ER subtypes measured in the solid-phase ligand-binding
system, are largely confirmed in the solubilized receptor
ligand-binding system. Coumestrol binds to ER with an affinity about
3-fold less than that of E2 itself, which is in agreement
with previously described data (38). Coumestrol binds with
essentially the same affinity as E2 to ERß. The
approximately 20-fold difference in binding affinity of genistein
observed in the solid-phase assay (incubation at ambient temperature
instead of 6 C) is confirmed, although the relative binding affinity
compared with E2 is, especially for ERß, lower (RBA
= 87 in Table 2 vs. RBA = 13 in Fig. 2).
Receptor-binding affinity is a function of temperature and equilibrium
time, and for steroid receptors the time necessary for equilibration of
receptor-radioligand complexes in the presence of competitor may be up
to 1000 min at the lower temperature (58). Because both ligand-binding
systems used incubation times of 18–20 h, it is unlikely that this
apparent discrepancy is caused by lack of equilibration. For
naringenin, apigenin and kaempferol complete displacement of
radioligand from the ER protein could not be obtained (Fig. 2), and
the competition curves are nonparallel for the ER subtypes. This could
point to binding-site heterogeneity, but further investigations are
needed to clarify this point.
|
and ERß
Human embryonal kidney 293 cells were transiently cotransfected with a
luciferase enzyme reporter gene construct containing three copies of a
consensus ERE in front of a TATA-box, together with human ER or
human ERß expression plasmids. As shown in Fig. 3, E2-stimulated reporter
gene activity by ERß was lower when compared with activity obtained
by ER. Also, half-maximal activation (EC50) is reached
at a lower concentration of E2 for ER than for ERß
(about 5 pM and about 50 pM, respectively). The
fold induction was relatively high, and therefore this transactivation
assay using embryonal kidney cells was considered to be very suitable
to estimate the estrogenic activity of compounds with low binding
affinity.
|
the (maximal) transcriptional
activity at a relatively high concentration of 1000 nM is
shown, while in Fig. 4 the
transcriptional activity is shown at various concentrations as
percentage of the maximal induction by E2 for each ER
subtype separately. The measured relative transactivation activities of
the suspected endocrine disruptors (Table 3 and Fig. 4) are comparable
with results from the radioligand competition assays, which showed
affinities up to 10,000-fold lower than E2. The OH-PCB-D
and OH-PCB-E compounds, which have a very low binding affinity
for both ER subtypes (Table 1) do not display any agonist activity.
Also, no antagonist activity could be detected in experiments with
various E2 concentrations and up to a 1000-fold excess of
OH-PCB-D or OH-PCB-E (not shown). On the other hand, the OH-PCB-K and
OH-PCB-L compounds, which have a higher binding affinity (Table 1), are
relatively strong agonists for both ER subtypes. With regard to the
organochlorine insecticides, the lack of significant binding affinity
of methoxychlor, endosulfan and p, p'-DDT is consistent with their low
agonist activities. Chlordecone (Kepone) is a weak agonist for ER,
but it has no agonist activity on ERß despite the fact that the
binding affinities are similar (Table 1). Neither on ERß nor on ER
any antagonist activity of chlordecone could be detected in experiments
in which up to a 10,000-fold excess of chlordecone was incubated
together with E2 (not shown). Bisphenol A is an equally
strong agonist for ER as for ERß, and the same is true for
4,4'-biphenol, which differs from bisphenol A in that it lacks the
propane group between the phenolic rings. No agonist activity of the
antiestrogens tamoxifen and ICI-182780 could be detected on ERß,
whereas tamoxifen had some agonistic activity on ER (Table 3).
Transcriptional stimulation observed for suspected endocrine disruptors
was dependent on cotransfected ER or ERß, confirming that the
transcriptional activation was mediated by the estrogen receptor (not
shown).
|
|
and ERß and Fig. 4)
was measured after incubation of transfected cell cultures with
concentrations of up to 1000 nM. In humans, peak serum
concentrations of total daidzein and total genistein of 500-1000
nM can be reached after consumption of meals rich in
soybeans or soybean protein extracts (41, 59). The phytoestrogens with
binding affinities 10,000-fold or more less than E2
(formononetin, ipriflavone, chrysin, quercetin) have very low or no
agonistic activity. Also, the binding affinity of biochanin A is more
than 10,000-fold less than E2 for both ER subtypes (Table 2); however, it has relatively strong agonistic activity (Table 3).
Biochanin A is the 4'-methylether of genistein, and it has been shown
that MCF-7 breast tumors cells can convert biochanin A to genistein
(60). A similar partial conversion of biochanin A to genistein by the
293 embryonal kidney cell line used for the transactivation assay might
explain the observed discrepancy. The estrogenic potency of the
remaining flavonoids (daidzein, apigenin, kaempferol, naringenin,
phloretin) at a concentration of 1000 nM is in line with
the observed 100- to 500-fold lower binding affinities for both ER
subtypes. Based upon these data (Fig. 4) and additional dose-response
curves not shown, a ranking of the estrogenic potencies of the
phytoestrogens is as follows: 17ß-estradiol >> zearalenone =
coumestrol > genistein > daidzein > apigenin =
phloretin > (biochanin A) = kaempferol = naringenin >
formononetin = ipriflavone = quercetin = chrysin for
ER and 17ß-estradiol >> genistein = coumestrol >
zearalenone > daidzein > (biochanin A) = apigenin =
kaempferol = naringenin > phloretin = quercetin =
ipriflavone = formononetin = chrysin for ERß. Although
these phytoestrogens are clearly less potent at inducing a biological
response than E2, some of them (genistein, zearalenone,
coumestrol) are able to generate a response of the same or almost the
same magnitude as that produced by the physiological hormone at
concentrations of 10–100 nM. In fact, at high
concentrations (1000 nM) the estrogenic potency of
genistein was greater than that of E2.
For zearalenone, antagonistic activity could be detected during
incubation of ERß transfected cell cultures with 1 nM
E2 and 100- to 1000-fold excess zearalenone. No
antagonistic activity of zearalenone could be detected when cell
cultures were transfected with ER (Fig. 5). In fact, zearalenone is a full
agonist for ER and a mixed agonist-antagonist for ERß in this
transactivation assay system (Fig. 5). For genistein (Fig. 5) and the
other phytoestrogens, no antagonism could be detected. Genistein and
coumestrol are full agonists on ER as well as ERß, although weaker
than E2 (Fig. 5). The half maximal activity for genistein
(Fig. 4) on ER is reached at about 20 nM (compared with
about 0.005 nM for E2) and for ERß at about 6
nM (compared with about 0.05 nM for
E2). Therefore, although the 20-fold higher binding
affinity of genistein for ERß (Table 2) is reflected in only a 3-fold
lower EC50 value, the relative estrogenic potency of
genistein on ERß is about 30-fold higher compared with the potency on
ER (estrogenic potency 0.005/20 x 100 = 0.025 for ER
and 0.05/6 x 100 = 0.8 for ERß with E2 = 100).
Similar calculations for coumestrol (Fig. 4) reveal an estrogenic
potency of 0.05 for ER and 0.5 for ERß with E2 = 100.
So, the higher binding affinity of coumestrol and genistein for ERß
is reflected in a clearly higher estrogenic potency. The
transcriptional activity of the phytoestrogens was dependent on
cotransfected ER or ERß expression plasmids, confirming that the
transcriptional activity was mediated by the estrogen receptor protein
(not shown).
|
|
Discussion |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
subtype but also
for the ERß subtype. Binding studies have provided a description of
the ligand structure-estrogen receptor binding affinity relationships
and a model for the ligand binding site (61). This model indicated that
the whole E2 skeleton, that is; the aromatic A-ring, the B-
and C-rings, and the OH-group in the D-ring contribute significantly to
receptor binding. It was also predicted that the receptor-bound ligand
is completely surrounded by the receptor with minimal exposure to
solvent. The recently determined crystal structure of the ER
ligand-binding domain complexed with E2 provided important
confirmation for this model (62). The phenolic hydroxyl group of the
A-ring of E2 nestles between two -helices and makes
several direct hydrogen bonds. This pincer-like arrangement around the
A-ring imposes an absolute requirement on ligands to contain an
aromatic ring, whereas the remainder of the binding pocket can accept a
number of different hydrophobic groups. The overall promiscuity of the
ER can be attributed to the size of the binding cavity, which has a
volume almost twice that of the E2 molecular volume. The
length and the width of the E2 skeleton is very well
matched by the receptor, but there are large unoccupied cavities
opposite the B-ring and the C-ring of E2 (62). Obviously,
several phytoestrogens (coumestrol, genistein) fit very well into the
available space, certainly for the ERß protein. It is difficult to
understand why other phytoestrogens do not exhibit higher binding
affinities because the orientation of the nonsteroidal ligands within
the binding pocket is unknown.
Although most of the estrogenic chemicals examined in this study
contain at least one aromatic ring with a hydroxyl group, their
relative affinities are generally 1000- to 10,000-fold lower than
E2. The complexes formed with the ER are probably very
unstable, as shown for various alkylphenols (63), and it is likely that
these compounds do not completely enter the ligand-binding pocket. The
observed radioligand competition might reflect blockade of
E2 entrance to the binding site or interaction with another
low affinity site that causes a change in the high affinity
E2 binding site. If this is true, it will be difficult to
use quantitative-structure activity relationship (QSAR) models
developed using ligands that bind with high affinity to predict those
chemical structures from compound libraries that might disrupt
development and reproduction in wildlife, as has been proposed recently
(64). Despite their very low binding affinities, several of the
suspected endocrine disruptors exhibit estrogenic activities in the
transactivation assay system with ER as well as ERß, albeit only
at a potency that is more than 1000-fold lower than that of
E2. Obviously, these compounds can induce at least
partially the conformational changes involved in the formation of a
transcriptionally competent activation function in the ligand-binding
domain (62). No striking differences in the relative binding affinities
for the tested compounds between ER and ERß could be detected.
Both ER subtypes could therefore be involved in the described
developmental and reproductive effects of estrogenic chemicals,
depending on their fetal tissue distribution pattern (17, 18, 19, 20, 21, 22, 30, 31, 32, 33, 34, 35).
The relatively low estrogenic potencies of suspected endocrine
disruptors suggests that these chemicals alone are unlikely to produce
adverse effects during fetal development (21). These compounds occur as
mixtures in the environment and diet, and synergistic transcriptional
activation of binary mixtures of weakly estrogenic chemicals have been
described (65). However, in subsequent detailed studies these
synergistic interactions for ER ligand-binding or transactivation could
not be confirmed (65, 66). Some suspected endocrine disruptors have
been shown to interact not only with the ER but also with the androgen
receptor or to interfere with steroid hormone synthesis or metabolism
(20). Combined effects of mixtures of endocrine disruptors with a
different mode of action could in this way result in synergistic
responses in vivo (20 and references therein). Most
suspected endocrine disruptors have been tested in in vitro
systems (radioligand competition, transactivation assays) and these
tests may underestimate or overestimate their in vivo
estrogenic potency. The estrogenic potency of bisphenol A in
vitro is 1000- to 5000-fold lower than that of E2, but
in vivo bisphenol A was rather effective in stimulating PRL
release from the pituitary (57). Development of in vivo
reporter systems for the assessment of the estrogenic activity of
suspected endocrine disruptors might be necessary. If the
ligand-binding domain of the ER is fused to a DNA-recombinase, the
recombinase activity is controlled efficiently by either agonistic or
antagonistic ligands (67, 68). Transgenic mice could be produced in
which activation of the recombinase hybrid is detected via elimination
of a disruption in a reporter gene (for instance galactosidase or lac
Z), thus enabling the use of a simple histochemical reaction in mouse
embryos to study the activity of suspected estrogenic chemicals. Of all
the suspected endocrine disruptors tested the OH-PCB-K and OH-PCB-L
compounds have the highest binding affinity (Table 1), but this is not
reflected in the transcription activation potency because compounds
with lower binding affinity have equally high estrogenic activity
(Table 1 and Table 3 and dose-response curves not shown). The
estrogenic potency of compounds is a complicated phenomenon that is the
result of a number of factors, such as differential effects on the
transactivation functionalities of the receptor, the particular
coactivators recruited and the cell- and target gene promoter-context
(62). The apparently lower transcriptional activity of ERß compared
with ER (Fig. 3) has also been reported in transient transfection
experiments using different cell lines (CHO, COS, HeLa) and reporter
gene constructs (11, 12, 13, 69). In contrast, in human osteosarcoma or
human endometrial carcinoma cells the transcriptional activity of ERß
was higher than that of ER (70). The reason for these differences in
transcriptional activity of the ER subtypes is at the moment unknown,
but it might reflect differential expression of transcriptional
coactivators or differential stability of the receptor proteins.
Several phytoestrogens have a higher binding affinity for the ERß
protein (Fig. 2), and both ER subtype transcripts are present in
prostate and breast tumor biopsies, although expression levels vary
widely (14, 71). In several epidemiological studies, an inverse
relation has been suggested between the risk of prostate cancer or
breast cancer and the intake of soy foods or the urinary excretion of
phytochemicals (39, 40, 41, 72, 73, 74), although in other studies this could
not be confirmed (72). The possibility still exists that the
association between reduced breast- and prostate cancer risk and
phytoestrogen intake is not causal, and merely results from some other
dietary characteristic. Despite the inconclusive epidemiological
findings, several putative mechanisms that could account for the
hypothesized chemopreventive effects of phytoestrogens have been
proposed. Most prominently, phytoestrogens have been suggested to exert
strong antiestrogenic effects, thereby inhibiting development of
hormone-related cancers (39, 72). In our study, only zearalenone
exhibited some antagonistic activity. All other phytoestrogens,
including the flavonoids that are present in soy foods, showed only
agonistic activity. In previous in vitro studies, involving
ER, only agonistic or at best partial antagonistic activities
instead of complete antagonistic activities were reported (36, 37, 38, 75).
Several other mechanisms for the proposed chemopreventive effects of
flavonoids have been suggested, including induction of cancer cell
differentiation, inhibition of protein tyrosine kinases,
suppression of angiogenesis, and direct antioxidant effects (41, 76).
These alternative mechanisms generally occur at flavonoid
concentrations much higher (>5 µM) than the
concentrations at which estrogenic effects are detected (<100
nM), and show a different structure-activity relationship;
moreover, the effects are observed in cells in the absence of ER
expression, and therefore it seems unlikely that all of these effects
are ER mediated (41, 77, 78). On the other hand, because both ER
subtypes are expressed in bone and the cardiovascular system (4, 79, 80, 81) and given the quite strong estrogenic activity of certain
phytoestrogens, the potential beneficial effects of increased food
intake of phytoestrogens in the prevention of postmenopausal
osteoporosis and cardiovascular diseases should be further investigated
(82).
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Supported in part by the European Union (EU-PL95–1223) Climate and
Environment program.
3 Supported in part by the Swedish Cancer Society and the European
Union (EU-PL95–1223) Climate and Environment program.
Received January 2, 1998.
|
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 A. M. Sotoca Covaleda, H. van den Berg, J. Vervoort, P. van der Saag, A. Strom, J.-A. Gustafsson, I. Rietjens, and A. J. Murk Influence of Cellular ER{alpha}/ER{beta} Ratio on the ER{alpha}-Agonist Induced Proliferation of Human T47D Breast Cancer Cells Toxicol. Sci., October 1, 2008; 105(2): 303 - 311. [Abstract] [Full Text] [PDF]
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Y. Hong, T. Wang, C. Huang, W. Cheng, and B. Lin Soy isoflavones supplementation alleviates disease severity in autoimmune-prone MRL-lpr/lpr mice Lupus, September 1, 2008; 17(9): 814 - 821. [Abstract] [PDF]
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 K.-M. Suzuki, Y. Isohama, H. Maruyama, Y. Yamada, Y. Narita, S. Ohta, Y. Araki, T. Miyata, and S. Mishima Estrogenic Activities of Fatty Acids and a Sterol Isolated from Royal Jelly Evid. Based Complement. Altern. Med., September 1, 2008; 5(3): 295 - 302. [Abstract] [Full Text] [PDF]
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G. S Prins Endocrine disruptors and prostate cancer risk Endocr. Relat. Cancer, September 1, 2008; 15(3): 649 - 656. [Abstract] [Full Text] [PDF]
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 A. Orgaard and L. Jensen The Effects of Soy Isoflavones on Obesity Experimental Biology and Medicine, September 1, 2008; 233(9): 1066 - 1080. [Abstract] [Full Text] [PDF]
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 N. Honma, R. Horii, T. Iwase, S. Saji, M. Younes, K. Takubo, M. Matsuura, Y. Ito, F. Akiyama, and G. Sakamoto Clinical Importance of Estrogen Receptor-{beta} Evaluation in Breast Cancer Patients Treated With Adjuvant Tamoxifen Therapy J. Clin. Oncol., August 1, 2008; 26(22): 3727 - 3734. [Abstract] [Full Text] [PDF]
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X. Di, L. Yu, A.B. Moore, L. Castro, X. Zheng, T. Hermon, and D. Dixon A low concentration of genistein induces estrogen receptor-alpha and insulin-like growth factor-I receptor interactions and proliferation in uterine leiomyoma cells Hum. Reprod., August 1, 2008; 23(8): 1873 - 1883. [Abstract] [Full Text] [PDF]
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 C. M. Klinge, N. S. Wickramasinghe, M. M. Ivanova, and S. M. Dougherty Resveratrol stimulates nitric oxide production by increasing estrogen receptor {alpha}-Src-caveolin-1 interaction and phosphorylation in human umbilical vein endothelial cells FASEB J, July 1, 2008; 22(7): 2185 - 2197. [Abstract] [Full Text] [PDF]
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P. Galluzzo, P. Ascenzi, P. Bulzomi, and M. Marino The Nutritional Flavanone Naringenin Triggers Antiestrogenic Effects by Regulating Estrogen Receptor {alpha}-Palmitoylation Endocrinology, May 1, 2008; 149(5): 2567 - 2575. [Abstract] [Full Text] [PDF]
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M. Hedelin, M. Lof, M. Olsson, H. Adlercreutz, S. Sandin, and E. Weiderpass Dietary Phytoestrogens Are Not Associated with Risk of Overall Breast Cancer But Diets Rich in Coumestrol Are Inversely Associated with Risk of Estrogen Receptor and Progesterone Receptor Negative Breast Tumors in Swedish Women J. Nutr., May 1, 2008; 138(5): 938 - 945. [Abstract] [Full Text] [PDF]
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M. Iwasaki, M. Inoue, T. Otani, S. Sasazuki, N. Kurahashi, T. Miura, S. Yamamoto, and S. Tsugane Plasma Isoflavone Level and Subsequent Risk of Breast Cancer Among Japanese Women: A Nested Case-Control Study From the Japan Public Health Center-Based Prospective Study Group J. Clin. Oncol., April 1, 2008; 26(10): 1677 - 1683. [Abstract] [Full Text] [PDF]
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R. Dip, S. Lenz, J.-P. Antignac, B. Le Bizec, H. Gmuender, and H. Naegeli Global gene expression profiles induced by phytoestrogens in human breast cancer cells Endocr. Relat. Cancer, March 1, 2008; 15(1): 161 - 173. [Abstract] [Full Text] [PDF]
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J. Mardon, J. Mathey, S. Kati-Coulibaly, C. Puel, M.-J. Davicco, P. Lebecque, M.-N. Horcajada, and V. Coxam Influence of Lifelong Soy Isoflavones Consumption on Bone Mass in the Rat Experimental Biology and Medicine, February 1, 2008; 233(2): 229 - 237. [Abstract] [Full Text] [PDF]
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 C. Bredhult, L. Sahlin, and M. Olovsson Gene expression analysis of human endometrial endothelial cells exposed to op'-DDT Mol. Hum. Reprod., February 1, 2008; 14(2): 97 - 106. [Abstract] [Full Text] [PDF]
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L. Lehmann, L. Jiang, and J. Wagner Soy isoflavones decrease the catechol-O-methyltransferase-mediated inactivation of 4-hydroxyestradiol in cultured MCF-7 cells Carcinogenesis, February 1, 2008; 29(2): 363 - 370. [Abstract] [Full Text] [PDF]
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H. Si and D. Liu Genistein, a Soy Phytoestrogen, Upregulates the Expression of Human Endothelial Nitric Oxide Synthase and Lowers Blood Pressure in Spontaneously Hypertensive Rats J. Nutr., February 1, 2008; 138(2): 297 - 304. [Abstract] [Full Text] [PDF]
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 A. Khan, R. G Berger, and D. deCatanzaro The onset of puberty in female mice as reflected in urinary steroids and uterine/ovarian mass: interactions of exposure to males, phyto-oestrogen content of diet, and ano-genital distance Reproduction, January 1, 2008; 135(1): 99 - 106. [Abstract] [Full Text] [PDF]
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 J. D. Gardner, G. L. Brower, T. G. Voloshenyuk, and J. S. Janicki Cardioprotection in female rats subjected to chronic volume overload: synergistic interaction of estrogen and phytoestrogens Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H198 - H204. [Abstract] [Full Text] [PDF]
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 J. P. Konhilas and L. A. Leinwand The Effects of Biological Sex and Diet on the Development of Heart Failure Circulation, December 4, 2007; 116(23): 2747 - 2759. [Full Text] [PDF]
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J. W. Lampe, Y. Nishino, R. M. Ray, C. Wu, W. Li, M.-G. Lin, D. L. Gao, Y. Hu, J. Shannon, H. Stalsberg, et al. Plasma Isoflavones and Fibrocystic Breast Conditions and Breast Cancer Among Women in Shanghai, China Cancer Epidemiol. Biomarkers Prev., December 1, 2007; 16(12): 2579 - 2586. [Abstract] [Full Text] [PDF]
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T. M. Cho, N. Peng, J. T. Clark, L. Novak, S. Roysommuti, J. Prasain, and J. M. Wyss Genistein Attenuates the Hypertensive Effects of Dietary NaCl in Hypertensive Male Rats Endocrinology, November 1, 2007; 148(11): 5396 - 5402. [Abstract] [Full Text] [PDF]
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 E. P. Moiseeva, G. M. Almeida, G. D.D. Jones, and M. M. Manson Extended treatment with physiologic concentrations of dietary phytochemicals results in altered gene expression, reduced growth, and apoptosis of cancer cells Mol. Cancer Ther., November 1, 2007; 6(11): 3071 - 3079. [Abstract] [Full Text] [PDF]
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 H. Malekinejad, E. J Schoevers, I. J.J.M Daemen, C. Zijlstra, B. Colenbrander, J. Fink-Gremmels, and B. A.J Roelen Exposure of Oocytes to the Fusarium Toxins Zearalenone and Deoxynivalenol Causes Aneuploidy and Abnormal Embryo Development in Pigs Biol Reprod, November 1, 2007; 77(5): 840 - 847. [Abstract] [Full Text] [PDF]
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 T. Sabo-Attwood, J. L Blum, K. J Kroll, V. Patel, D. Birkholz, N. J Szabo, S. Z Fisher, R. McKenna, M. Campbell-Thompson, and N. D Denslow Distinct expression and activity profiles of largemouth bass (Micropterus salmoides) estrogen receptors in response to estradiol and nonylphenol J. Mol. Endocrinol., October 1, 2007; 39(4): 223 - 237. [Abstract] [Full Text] [PDF]
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M. Chang, K.-w. Peng, I. Kastrati, C. R. Overk, Z.-H. Qin, P. Yao, J. L. Bolton, and G. R. J. Thatcher Activation of Estrogen Receptor-Mediated Gene Transcription by the Equine Estrogen Metabolite, 4-Methoxyequilenin, in Human Breast Cancer Cells Endocrinology, October 1, 2007; 148(10): 4793 - 4802. [Abstract] [Full Text] [PDF]
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P. Penttinen, J. Jaehrling, A. E. Damdimopoulos, J. Inzunza, J. G. Lemmen, P. van der Saag, K. Pettersson, G. Gauglitz, S. Makela, and I. Pongratz Diet-Derived Polyphenol Metabolite Enterolactone Is a Tissue-Specific Estrogen Receptor Activator Endocrinology, October 1, 2007; 148(10): 4875 - 4886. [Abstract] [Full Text] [PDF]
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S. Ray, F. Xu, P. Li, N. S. Sanchez, H. Wang, and S. K. Das Increased Level of Cellular Bip Critically Determines Estrogenic Potency for a Xenoestrogen Kepone in the Mouse Uterus Endocrinology, October 1, 2007; 148(10): 4774 - 4785. [Abstract] [Full Text] [PDF]
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A.B. Moore, L. Castro, L. Yu, X. Zheng, X. Di, M.I. Sifre, G.E. Kissling, R.R. Newbold, C.D. Bortner, and D. Dixon Stimulatory and inhibitory effects of genistein on human uterine leiomyoma cell proliferation are influenced by the concentration Hum. Reprod., October 1, 2007; 22(10): 2623 - 2631. [Abstract] [Full Text] [PDF]
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S. Bolca, S. Possemiers, A. Herregat, I. Huybrechts, A. Heyerick, S. De Vriese, M. Verbruggen, H. Depypere, D. De Keukeleire, M. Bracke, et al. Microbial and Dietary Factors Are Associated with the Equol Producer Phenotype in Healthy Postmenopausal Women J. Nutr., October 1, 2007; 137(10): 2242 - 2246. [Abstract] [Full Text] [PDF]
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M. Stettner, S. Kaulfuss, P. Burfeind, S. Schweyer, A. Strauss, R.-H. Ringert, and P. Thelen The relevance of estrogen receptor-{beta} expression to the antiproliferative effects observed with histone deacetylase inhibitors and phytoestrogens in prostate cancer treatment Mol. Cancer Ther., October 1, 2007; 6(10): 2626 - 2633. [Abstract] [Full Text] [PDF]
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 C. Duffy, K. Perez, and A. Partridge Implications of Phytoestrogen Intake for Breast Cancer CA Cancer J Clin, September 1, 2007; 57(5): 260 - 277. [Abstract] [Full Text] [PDF]
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A. Beleza-Meireles, I. Kockum, F. Lundberg, C. Soderhall, and A. Nordenskjold Risk Factors for Hypospadias in the Estrogen Receptor 2 Gene J. Clin. Endocrinol. Metab., September 1, 2007; 92(9): 3712 - 3718. [Abstract] [Full Text] [PDF]
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 M. Subramanian and C. Shaha Up-Regulation of Bcl-2 through ERK Phosphorylation Is Associated with Human Macrophage Survival in an Estrogen Microenvironment J. Immunol., August 15, 2007; 179(4): 2330 - 2338. [Abstract] [Full Text] [PDF]
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M. Yoshioka, A. Boivin, C. Bolduc, and J. St-Amand Gender difference of androgen actions on skeletal muscle transcriptome J. Mol. Endocrinol., August 1, 2007; 39(2): 119 - 133. [Abstract] [Full Text] [PDF]
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M. Atteritano, H. Marini, L. Minutoli, F. Polito, A. Bitto, D. Altavilla, S. Mazzaferro, R. D'Anna, M. L. Cannata, A. Gaudio, et al. Effects of the Phytoestrogen Genistein on Some Predictors of Cardiovascular Risk in Osteopenic, Postmenopausal Women: A Two-Year Randomized, Double-Blind, Placebo-Controlled Study J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3068 - 3075. [Abstract] [Full Text] [PDF]
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Y. Chen, W. N. Jefferson, R. R. Newbold, E. Padilla-Banks, and M. E. Pepling Estradiol, Progesterone, and Genistein Inhibit Oocyte Nest Breakdown and Primordial Follicle Assembly in the Neonatal Mouse Ovary in Vitro and in Vivo Endocrinology, August 1, 2007; 148(8): 3580 - 3590. [Abstract] [Full Text] [PDF]
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 E. Murphy and C. Steenbergen Gender-based differences in mechanisms of protection in myocardial ischemia-reperfusion injury Cardiovasc Res, August 1, 2007; 75(3): 478 - 486. [Abstract] [Full Text] [PDF]
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 H. Marini, L. Minutoli, F. Polito, A. Bitto, D. Altavilla, M. Atteritano, A. Gaudio, S. Mazzaferro, A. Frisina, N. Frisina, et al. Effects of the Phytoestrogen Genistein on Bone Metabolism in Osteopenic Postmenopausal Women: A Randomized Trial Ann Intern Med, June 19, 2007; 146(12): 839 - 847. [Abstract] [Full Text] [PDF]
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A. M Dorward, K. L Shultz, and W. G Beamer LH analog and dietary isoflavones support ovarian granulosa cell tumor development in a spontaneous mouse model Endocr. Relat. Cancer, June 1, 2007; 14(2): 369 - 379. [Abstract] [Full Text] [PDF]
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M. Penza, C. Montani, A. Romani, P. Vignolini, P. Ciana, A. Maggi, B. Pampaloni, L. Caimi, and D. Di Lorenzo Genistein Accumulates in Body Depots and Is Mobilized during Fasting, Reaching Estrogenic Levels in Serum that Counter the Hormonal Actions of Estradiol and Organochlorines Toxicol. Sci., June 1, 2007; 97(2): 299 - 307. [Abstract] [Full Text] [PDF]
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J. L. Kipp, S. M. Kilen, S. Bristol-Gould, T. K. Woodruff, and K. E. Mayo Neonatal Exposure to Estrogens Suppresses Activin Expression and Signaling in the Mouse Ovary Endocrinology, May 1, 2007; 148(5): 1968 - 1976. [Abstract] [Full Text] [PDF]
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T. Tiido, A. Rignell-Hydbom, B.A.G. Jonsson, L. Rylander, A. Giwercman, and Y.L. Giwercman Modifying effect of the AR gene trinucleotide repeats and SNPs in the AHR and AHRR genes on the association between persistent organohalogen pollutant exposure and human sperm Y : X ratio Mol. Hum. Reprod., April 1, 2007; 13(4): 223 - 229. [Abstract] [Full Text] [PDF]
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 S. K. Gruvberger-Saal, P.-O. Bendahl, L. H. Saal, M. Laakso, C. Hegardt, P. Eden, C. Peterson, P. Malmstrom, J. Isola, A. Borg, et al. Estrogen Receptor {beta} Expression Is Associated with Tamoxifen Response in ER{alpha}-Negative Breast Carcinoma Clin. Cancer Res., April 1, 2007; 13(7): 1987 - 1994. [Abstract] [Full Text] [PDF]
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 E. T. Chang, V. S. Lee, A. J. Canchola, C. A. Clarke, D. M. Purdie, P. Reynolds, H. Anton-Culver, L. Bernstein, D. Deapen, D. Peel, et al. Diet and Risk of Ovarian Cancer in the California Teachers Study Cohort Am. J. Epidemiol., April 1, 2007; 165(7): 802 - 813. [Abstract] [Full Text] [PDF]
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 M. S. Touillaud, A. C. M. Thiebaut, A. Fournier, M. Niravong, M.-C. Boutron-Ruault, and F. Clavel-Chapelon Dietary Lignan Intake and Postmenopausal Breast Cancer Risk by Estrogen and Progesterone Receptor Status J Natl Cancer Inst, March 21, 2007; 99(6): 475 - 486. [Abstract] [Full Text] [PDF]
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Y.-C. Chen, M. L Nagpal, D. M Stocco, and T. Lin Effects of genistein, resveratrol, and quercetin on steroidogenesis and proliferation of MA-10 mouse Leydig tumor cells J. Endocrinol., March 1, 2007; 192(3): 527 - 537. [Abstract] [Full Text] [PDF]
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A. L. Quinn, J. M. Regan, J. M. Tobin, B. J. Marinik, J. M. McMahon, D. A. McNett, C. M. Sushynski, S. D. Crofoot, P. A. Jean, and K. P. Plotzke In Vitro and In Vivo Evaluation of the Estrogenic, Androgenic, and Progestagenic Potential of Two Cyclic Siloxanes Toxicol. Sci., March 1, 2007; 96(1): 145 - 153. [Abstract] [Full Text] [PDF]
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M. Verheus, C. H. van Gils, L. Keinan-Boker, P. B. Grace, S. A. Bingham, and P. H.M. Peeters Plasma Phytoestrogens and Subsequent Breast Cancer Risk J. Clin. Oncol., February 20, 2007; 25(6): 648 - 655. [Abstract] [Full Text] [PDF]
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A. Cvoro, S. Paruthiyil, J. O. Jones, C. Tzagarakis-Foster, N. J. Clegg, D. Tatomer, R. T. Medina, M. Tagliaferri, F. Schaufele, T. S. Scanlan, et al. Selective Activation of Estrogen Receptor-{beta} Transcriptional Pathways by an Herbal Extract Endocrinology, February 1, 2007; 148(2): 538 - 547. [Abstract] [Full Text] [PDF]
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B. K. Chacko, R. T. Chandler, T. L. D'Alessandro, A. Mundhekar, N. K. H. Khoo, N. Botting, S. Barnes, and R. P. Patel Anti-Inflammatory Effects of Isoflavones are Dependent on Flow and Human Endothelial Cell PPAR{gamma} J. Nutr., February 1, 2007; 137(2): 351 - 356. [Abstract] [Full Text] [PDF]
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 H. A. Harris Estrogen Receptor-{beta}: Recent Lessons from in Vivo Studies Mol. Endocrinol., January 1, 2007; 21(1): 1 - 13. [Abstract] [Full Text] [PDF]
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 I. M. Medina-Diaz, G. Arteaga-Illan, M. B. de Leon, B. Cisneros, A. Sierra-Santoyo, L. Vega, F. J. Gonzalez, and G. Elizondo Pregnane X Receptor-Dependent Induction of the CYP3A4 Gene by o,p'-1,1,1,-Trichloro-2,2-Bis (p-Chlorophenyl)ethane Drug Metab. Dispos., January 1, 2007; 35(1): 95 - 102. [Abstract] [Full Text] [PDF]
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S. Assinder, R. Davis, M. Fenwick, and A. Glover Adult-only exposure of male rats to a diet of high phytoestrogen content increases apoptosis of meiotic and post-meiotic germ cells Reproduction, January 1, 2007; 133(1): 11 - 19. [Abstract] [Full Text] [PDF]
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L. N. Vandenberg, M. V. Maffini, P. R. Wadia, C. Sonnenschein, B. S. Rubin, and A. M. Soto Exposure to Environmentally Relevant Doses of the Xenoestrogen Bisphenol-A Alters Development of the Fetal Mouse Mammary Gland Endocrinology, January 1, 2007; 148(1): 116 - 127. [Abstract] [Full Text] [PDF]
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G. Bises, E. Bajna, T. Manhardt, W. Gerdenitsch, E. Kallay, and H. S. Cross Gender-Specific Modulation of Markers for Premalignancy by Nutritional Soy and Calcium in the Mouse Colon J. Nutr., January 1, 2007; 137(1): 211S - 215S. [Abstract] [Full Text] [PDF]
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S. Rice and S. A Whitehead Phytoestrogens and breast cancer -promoters or protectors? Endocr. Relat. Cancer, December 1, 2006; 13(4): 995 - 1015. [Abstract] [Full Text] [PDF]
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 K. Dahlman-Wright, V. Cavailles, S. A. Fuqua, V. C. Jordan, J. A. Katzenellenbogen, K. S. Korach, A. Maggi, M. Muramatsu, M. G. Parker, and J.-A. Gustafsson International Union of Pharmacology. LXIV. Estrogen Receptors Pharmacol. Rev., December 1, 2006; 58(4): 773 - 781. [Full Text] [PDF]
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M. Penza, C. Montani, A. Romani, P. Vignolini, B. Pampaloni, A. Tanini, M. L. Brandi, P. Alonso-Magdalena, A. Nadal, L. Ottobrini, et al. Genistein Affects Adipose Tissue Deposition in a Dose-Dependent and Gender-Specific Manner Endocrinology, December 1, 2006; 147(12): 5740 - 5751. [Abstract] [Full Text] [PDF]
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K. J. McInnes, A. Corbould, E. R. Simpson, and M. E. Jones Regulation of Adenosine 5',Monophosphate-Activated Protein Kinase and Lipogenesis by Androgens Contributes to Visceral Obesity in an Estrogen-Deficient State Endocrinology, December 1, 2006; 147(12): 5907 - 5913. [Abstract] [Full Text] [PDF]
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S. C. Laws, S Yavanhxay, R. L. Cooper, and J. C. Eldridge Nature of the Binding Interaction for 50 Structurally Diverse Chemicals with Rat Estrogen Receptors Toxicol. Sci., November 1, 2006; 94(1): 46 - 56. [Abstract] [Full Text] [PDF]
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 K. W. Wilhelms, C. G. Scanes, and L. L. Anderson Lack of estrogenic or antiestrogenic actions of soy isoflavones in an avian model: the Japanese quail. Poult. Sci., November 1, 2006; 85(11): 1885 - 1889. [Abstract] [Full Text] [PDF]
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M. Raschke, I. R. Rowland, P. J. Magee, and B. L. Pool-Zobel Genistein protects prostate cells against hydrogen peroxide-induced DNA damage and induces expression of genes involved in the defence against oxidative stress Carcinogenesis, November 1, 2006; 27(11): 2322 - 2330. [Abstract] [Full Text] [PDF]
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A. R. Thomsen, K. Almstrup, J. E. Nielsen, I. K. Sorensen, O. W. Petersen, H. Leffers, and V. M. Breinholt Estrogenic Effect of Soy Isoflavones on Mammary Gland Morphogenesis and Gene Expression Profile Toxicol. Sci., October 1, 2006; 93(2): 357 - 368. [Abstract] [Full Text] [PDF]
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C. Borras, J. Gambini, M. C. Gomez-Cabrera, J. Sastre, F. V. Pallardo, G. E. Mann, and J. Vina Genistein, a soy isoflavone, up-regulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NF{kappa}B FASEB J, October 1, 2006; 20(12): 2136 - 2138. [Abstract] [Full Text] [PDF]
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 C. Bolego, E. Vegeto, C. Pinna, A. Maggi, and A. Cignarella Selective Agonists of Estrogen Receptor Isoforms: New Perspectives for Cardiovascular Disease Arterioscler Thromb Vasc Biol, October 1, 2006; 26(10): 2192 - 2199. [Abstract] [Full Text] [PDF]
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; ERß =
) was calculated by subtracting nonspecific bound counts from total
bound counts. Inset, Scatchard plot analysis of specific
binding giving a Kd of 0.05 nM for ER
and ERß =
• or 
. Abscissa, log M of compound; ordinate, transcriptional
activity as percentage of the maximal induction by E2 for
each ER subtype.