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Bilimsel çalışma 1

Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor β

1. George G. J. M. Kuiper 1 ,

2. Josephine G. Lemmen,

3. Bo Carlsson,

4. J. Christopher Corton,

5. Stephen H. Safe,

6. Paul T. van der Saag,

7. Bart van der Burg 2 and

8. Jan-Åke Gustafsson 3

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@csb.ki.se.

Abstract

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ERα and ERβ, 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 ERα or ERβ protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ERα or ERβ complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid.

Saturation ligand-binding analysis of human ERα and ERβ protein revealed a single binding component for[ 3H]-17β-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 ERβ than to ERα. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p′-DDT and 2′,4′,6′-trichloro-4-biphenylol stimulate the transcriptional activity of ERα and ERβ 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 ERα and E2 ≫ genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ERβ. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ERα and a mixed agonist-antagonist for ERβ. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ERβ, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

THE STEROID hormone estrogen influences the growth, differentiation, and functioning of many target tissues. These include tissues of the female and male reproductive systems such as mammary gland, uterus, vagina, ovary, testes, epididymis, and prostate (1). Estrogens also play an important role in bone maintenance, in the central nervous system and in the cardiovascular system where estrogens have certain cardioprotective effects (1, 2, 3, 4). Estrogens diffuse in and out of cells but are retained with high affinity and specificity in target cells by an intranuclear binding protein, termed the estrogen receptor (ER). Once bound by estrogens, the ER undergoes a conformational change allowing the receptor to interact with chromatin and to modulate transcription of target genes (5, 6, 7). We have cloned a novel ER cDNA from rat prostate (8), named ERβ, different from the previously cloned ER cDNA (consequently renamed ERα). Rat ERβ cDNA encodes a protein of 485 amino acid residues with a calculated molecular weight of 54200. Rat ERβ protein is highly homologous to rat ERα 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 ERβ 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 ERβ have been cloned, and similar homologies in the various domains of the subtypes were found. Expression of ERβ 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 17β-estradiol (E2) by ERβ protein, and ERβ 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 ERα vs. ERβ, 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 ERα protein extracted from rat uterus or human breast tumor cells or with recombinant ERα protein (23, 24, 25, 26, 27, 28, 29). No data are yet available on the potential interaction of estrogenic chemicals with ERβ, and interactions of xenoestrogens with this subtype may be related to some recent observations. In the rat and mouse prostate, ERβ 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, ERβ 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 ERβ 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 17β-estradiol for binding to ERα protein, and induce activity of estrogen-responsive reporter gene constructs in the presence of ERα 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 ERβ 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 ERα or ERβ protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ERα or ERβ cDNA in the presence of an estrogen-dependent reporter plasmid.

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Materials and Methods

Materials

The steroids 17β-estradiol, 17α-estradiol (1, 3, 5(10)-estratriene-3,17α-diol), 16-keto-17β-estradiol (1, 3, 5(10)-estratriene-3,17β-diol-16-one), 17-epiestriol (1, 3, 5(10)-estratriene-3,16α,17α-triol), 16α-bromoestradiol (1, 3, 5(10)-estratriene-16α-bromo-3,17β-diol), 2-OH-estrone (1, 3, 5(10)-estratriene-2,3-diol-17-one), progesterone, 5-androstenediol (5-androstene-3β, 17β-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-β-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, 17β-dihydroxyestra-1, 3, 5(10)trien-7α-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 (Tyresō, 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 ERα and ERβ protein extracts

A 1.5-kb DNA fragment encoding the human homolog of rat ERβ protein (485 amino acid residues) was excised with SacII/SpeI from pGEM-T/hERβ (14) and isolated from agarose gel. The fragment was ligated to a BamHI/SacII adapter, recut with BamHI/SpeI and ligated into theBamHI/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 ERα coding sequence was derived from the mammalian expression vector pMT-hERα. The plasmid was linearized with SacI, and a BamHI linker was ligated after T4 DNA-polymerase treatment. The 1.9-kb fragment encoding hERα 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/hERα 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-17β-estradiol binding with the solubilized receptor based assay (see below). The ERα extract contained 400 pmol receptor/ml and the ERβ 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 (ERα extract 50-fold and ERβ 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-17β-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 mMEDTA, 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-17β-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; 1μ l/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-17β-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 (3×ERE-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 ERβ expression plasmid pSG5-hERβ contains a 1.5 kb human ERβ cDNA, encoding the 485 amino acid residue human ERβ protein as described (14). The human ERα 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 1μ g 3×ERE-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 theβ -galactosidase activity was measured to correct for variations in transfection efficiencies (51).

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Results

Expression and saturation ligand binding analysis of ER protein

Various steroid receptors including human ERα 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 ERα and ERβ protein expressed in insect cells for the ligand binding experiments.

In Fig. 1⇓, the result of a saturation ligand binding experiment with [3H]-17β-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 ERα and 0.07 nM for ERβ protein. Linear transformation of saturation data (Scatchard plots in Fig. 1⇓) revealed a single population of binding sites for 17β-estradiol with a Kd of 0.05 nMfor the ERα protein and 0.09 nM for ERβ protein. In a previous report (15) we found a 4-fold higher affinity for ERα compared with ERβ, however, in that study 16α-[125I]-iodo-17β-estradiol was used as ligand instead of [3H]-17β-estradiol.

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Figure 1.

Binding of 3H-17β-estradiol to recombinant ERα and ERβ protein (solubilized receptor assay) in the presence or absence of a 300-fold excess of E2 for 18 h at 6 C. Unbound radioligand was removed as described (solubilized receptor assay), and specific bound radioligand (ERα = ○; ERβ =□ ) 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 ERα protein and a Kd of 0.09 nM for ERβ protein.

Ligand binding specificity of ERα and ERβ protein

Measurements of the equilibrium binding of the radioligand in the presence of different concentrations of unlabeled competitors provide readily interpretable information about the affinities of the latter. To group a large number of suspected endocrine disruptors and phytoestrogens into those which show significant affinity for both ER subtypes and those which do not bind at all, we used a previously developed solid-phase binding system as a screening assay (45). In the solid-phase binding assay recombinantly produced human ERα and ERβ 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 ERα and ERβ 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 16α-bromo-17β-estradiol for ERα is in line with the measured 4-fold higher Kd (= lower affinity) of ERβ compared with ERα for the radioligand 16α-iodo-17β-estradiol (15). The selective estrogen receptor modulator (SERM) raloxifene and various E2 metabolites (17-epiestriol and 16-keto-17β-estradiol) that have been shown to stimulate ERα mediated TGF-β3 gene transcription in bone cells via a novel non-ERE-dependent pathway (54), also interact with the ERβ protein.

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Table 1.

RBA of suspected environmental endocrine disruptors for ERα and ERβ from solid-phase (Scintistrip) competition experiments

Several suspected endocrine disruptors bind weakly to ERα and ERβ protein

The environmental estrogen o, p′-DDT binds weakly to ERα (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 ERα and ERβ 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 ERα as well as ERβ 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 ERα (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 ERβ 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 ERα and ERβ.

Differential binding of several phytoestrogens to ERα and ERβ protein

The binding affinity of coumestrol to ERβ is 7-fold higher in comparison to ERα, 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 ERβ 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 ERβ, 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.

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Table 2.

Binding affinity of various phytoestrogens for ERα and ERβ

To confirm the sometimes quite large differences in relative binding affinity determined in the solid-phase ligand-binding system (Table 2⇑), 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 16α-bromo-17β-estradiol was significantly higher (about 4-fold) for ERα, whereas the binding affinity of 5-androstenediol is significantly higher for ERβ, 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 ERα 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 ERβ. 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 ERβ, 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 ERα 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.

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Figure 2.

Competition (solubilized receptor assay) by several nonradioactive (phyto)-estrogens and antiestrogens for 3H-17β-estradiol binding to ERα (○) and ERβ protein (□). Incubation was for 18 h at 6 C, and bound and unbound radioligand were separated as described for the solubilized receptor assay. Abscissa, log M of compound; ordinate, dpm bound radioligand.

Suspected endocrine disruptors stimulate the transcriptional activity of ERα and ERβ

In radioligand competition assays only compounds able to displace or compete with the radioligand for binding to the receptor are detected. Furthermore, ligand-binding assays do not disclose the biological activity of a compound, i.e whether it is an agonist or an antagonist. Animals have traditionally been used for the biological profiling of compounds; however, these assays are costly and time-consuming. An alternative for initial characterization of compounds is a cell based transcription assay system, using a reporter gene under the transcriptional control of a specific receptor.

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 ERα or human ERβ expression plasmids. As shown in Fig. 3⇓, E2-stimulated reporter gene activity by ERβ was lower when compared with activity obtained by ERα. Also, half-maximal activation (EC50) is reached at a lower concentration of E2 for ERα than for ERβ (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.

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Figure 3.

Activation of transcription by E2 in human embryonal kidney 293 cells. Cells were transfected with ERE-TATA-Luc reporter plasmid, and pSG5-hERα (○) or pSG5-hERβ (•) expression plasmid. After 16 h, the medium was changed and E2 or vehicle was added (c = control). After 24 h incubation, the cells were lysed and the reporter gene activity was measured. Results are expressed as fold induction ±SD from two different experiments with each concentration in triplicate.

To obtain an impression of the transcription stimulating activity of the compounds tested in the radioligand-competition assay, a selection of these compounds was tested at concentrations up to 1000 nM in the transactivation assay. It should be noted that in Table 3⇓ 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 ERα, but it has no agonist activity on ERβ despite the fact that the binding affinities are similar (Table 1⇑). Neither on ERβ nor on ERα 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 ERα as for ERβ, 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 ERβ, whereas tamoxifen had some agonistic activity on ERα (Table 3⇓). Transcriptional stimulation observed for suspected endocrine disruptors was dependent on cotransfected ERα or ERβ, confirming that the transcriptional activation was mediated by the estrogen receptor (not shown).

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Figure 4.

Activation of transcription by various estrogenic chemicals and phytoestrogens. The experiment (two different experiments with each point in triplicate) were done as described in Materials and Methods. ERα = ○ or ▵ and ERβ =• or ▴. Abscissa, log M of compound; ordinate, transcriptional activity as percentage of the maximal induction by E2 for each ER subtype.

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Table 3.

Relative transactivation activity1 of various compounds for ERα and ERβ

Flavonoids, coumestrol, and zearalenone stimulate transcriptional activity mediated by ERα and ERβ

Transactivation activity of phytoestrogens (Table 3⇑ 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: 17β-estradiol ≫ zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > (biochanin A) = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ERα and 17β-estradiol ≫ genistein = coumestrol > zearalenone > daidzein > (biochanin A) = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ERβ. 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 ERβ 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 ERα (Fig. 5⇓). In fact, zearalenone is a full agonist for ERα and a mixed agonist-antagonist for ERβ 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 ERα as well as ERβ, although weaker than E2 (Fig. 5⇓). The half maximal activity for genistein (Fig. 4⇑) on ERα is reached at about 20 nM (compared with about 0.005 nM for E2) and for ERβ at about 6 nM(compared with about 0.05 nM for E2). Therefore, although the 20-fold higher binding affinity of genistein for ERβ (Table 2⇑) is reflected in only a 3-fold lower EC50 value, the relative estrogenic potency of genistein on ERβ is about 30-fold higher compared with the potency on ERα (estrogenic potency 0.005/20 × 100 = 0.025 for ERα and 0.05/6 × 100 = 0.8 for ERβ with E2 = 100). Similar calculations for coumestrol (Fig. 4⇑) reveal an estrogenic potency of 0.05 for ERα and 0.5 for ERβ with E2 = 100. So, the higher binding affinity of coumestrol and genistein for ERβ is reflected in a clearly higher estrogenic potency. The transcriptional activity of the phytoestrogens was dependent on cotransfected ERα or ERβ expression plasmids, confirming that the transcriptional activity was mediated by the estrogen receptor protein (not shown).

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Figure 5.

Activation of transcription by zearalenone and genistein in the absence or presence of E2. A, Transfected cell-cultures were incubated (conc. shown as -log M) with zearalenone (ZEA), ICI-182780 (ICI) or E2alone or in combinations as indicated. Results are expressed as fold induction over vehicle only incubation ±SD for two different experiments with each combination in triplicate. B, Transfected cell-cultures were incubated (concentration shown as -log M) with genistein (GEN), ICI-182780 (ICI) or E2 alone and in combinations as indicated. Results are expressed as fold induction over vehicle only incubation± SD for two different experiments with each combination in triplicate.

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Discussion

The ER binds a large number of compounds that exhibit remarkably diverse structural features. In fact, the estrogen receptor is probably unique among the steroid receptors in its ability to interact with a wide variety of compounds. This is true for the ERα subtype but also for the ERβ 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 ERα 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 α-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 ERβ 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 ERα as well as ERβ, 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 ERα and ERβ 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 vitrosystems (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 ERβ compared with ERα (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 ERβ was higher than that of ERα (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 ERβ 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 vitrostudies, involving ERα, 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).

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Acknowledgments

We thank Tomas Barkhem and Birgitta Möller (KaroBio, Huddinge) for the preparation of insect cells expressing ER subtype proteins, Kevin Gaido (CIIT) for the preparation of some of the estrogenic chemicals, Sari Mäkelä (University of Turku, Finland) for providing several phytoestrogens, Alan Wakeling (Zeneca Pharmaceuticals, Macclesfield, Cheshire, UK) for providing ICI 182780 and Margaret Warner (Department of Medical Nutrition, Karolinska Institute) for helpful discussions and comments on the manuscript.

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Footnotes

• ↵1 Supported by the Swedish Medical Research Council (MFR K98–04P-12596–01A), and the Loo och Hans Ostermans Stiftelse.

• ↵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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Bilimsel çalışma 2

Isoﬂavone-Rich Soy Protein Isolate Suppresses

Androgen Receptor Expression without Altering

Estrogen Receptor-b Expression or Serum

Hormonal Proﬁles in Men at High Risk

of Prostate Cancer

1–3

Jill M. Hamilton-Reeves,

Salome A. Rebello,

William Thomas,

Joel W. Slaton,

6,7

and Mindy S. Kurzer

Department of Food Science and Nutrition;

Division of Biostatistics in the School of Public Health; and

Department of Urologic

Surgery, University of Minnesota, Minneapolis, MN 55455 and

Department of Urology, Veterans Administration Medical Center,

Minneapolis, MN 55417

Abstract

The purpose of this study was to determine the effects of soy protein isolate consumption on circulating hormone proﬁles

and hormone receptor expression patterns in men at high risk for developing advanced prostate cancer. Fifty-eight men

were randomly assigned to consume 1 of 3 protein isolates containing 40 g/d protein: 1) soy protein isolate (SPI1) (107

mg/d isoﬂavones); 2) alcohol-washed soy protein isolate (SPI2) (,6 mg/d isoﬂavones); or 3) milk protein isolate (0 mg/d

isoﬂavones). For 6 mo, the men consumed the protein isolates in divided doses twice daily as a partial meal replacement.

Serum samples collected at 0, 3, and 6 mo were analyzed for circulating estradiol, estrone, sex hormone-binding globulin,

androstenedione, androstanediol glucuronide, dehydroepiandrosterone sulfate, dihydrotestosterone, testosterone, and

free testosterone concentrations by RIA. Prostate biopsy samples obtained pre- and postintervention were analyzed

for androgen receptor (AR) and estrogen receptor-b expression by immunohistochemistry. At 6 mo, consumption of

SPI1 signiﬁcantly suppressed AR expression but did not alter estrogen receptor-b expression or circulating hormones.

Consumption of SPI2 signiﬁcantly increased estradiol and androstenedione concentrations, and tended to suppress AR

expression (P ¼ 0.09). Although the effects of SPI2 consumption on estradiol and androstenedione are difﬁcult to

interpret and the clinical relevance is uncertain, these data show that AR expression in the prostate is suppressed by soy

protein isolate consumption, which may be beneﬁcial in preventing prostate cancer. J. Nutr. 137: 1769–1775, 2007.

Introduction

Steroid hormones modulate growth of the prostate gland, and

elevated levels of androgens have been associated with prostate

cancer risk (1,2). Consumption of soy foods is thought to contribute to prostate cancer prevention as a result of the hormonal

properties of soy isoﬂavones, either through altered endogenous

circulating hormones or hormone-receptor signaling. Cell culture studies have suggested that the isoﬂavonoids, genistein and

equol, exert the most noteworthy hormonal effects. Genistein

inhibits the activity of 5a-reductase and 17b-hydroxysteroid dehydrogenase, enzymes required for androgen synthesis (3,4). The

isoﬂavonoid equol, a bacterially derived metabolite of the iso-

ﬂavone daidzein, sequesters dihydrotestosterone (DHT)

from

the androgen receptor (AR) in rat prostate tissue (5). Both isoﬂavonoids accumulate in the prostate gland (6–9) and may mimic or

modulate endogenous hormones relevant to prostate carcinogenesis.

Despite evidence from in vitro studies, human intervention

studies report inconsistent effects of soy or isoﬂavone consumption on circulating hormone proﬁles in men. Although reports

show statistically signiﬁcant suppression of total testosterone

(10,11), sex hormone binding globulin (SHBG) (12), DHT (13),

dehydroepiandrosterone (14), estrone (15), and free androgen

index (13), and increased concentrations of SHBG (16) and DHT

(17), the majority of the 22 intervention studies to date have not

found signiﬁcant changes in circulating sex steroid hormones (10–

31). Generally, the studies that report signiﬁcant changes were

The Soy and Prostate Cancer Prevention (SoyCaP) trial was supported by grant

DAMD 17-02-1-0101 (M.S.K.) and W81XWH-06-1-0075 (J.H.R.) from the United

States Army Department of Defense Prostate Cancer Research Program. The

protein isolates were donated by The Solae Company, St. Louis, MO. Neither

sponsor was involved in writing this report.

Author disclosures: J. M. Hamilton-Reeves, S. A. Rebello, W. Thomas, J. W.

Slaton, and M. S. Kurzer, no conﬂicts of interest.

Supplemental Tables 1 and 2 are available with the online posting of this paper

at jn.nutrition.org.

* To whom correspondence should be addressed. E-mail: mkurzer@umn.edu.

Abbreviations used: 3a-AG, androstanediol glucuronide; AR, androgen receptor; DHEAS, dehydroepiandrosterone sulfate; DHT, dihydrotestosterone; ERb,

estrogen receptor-b; MPI, milk protein isolate; SHBG, sex hormone-binding

globulin; SPI2, alcohol-extracted soy protein isolate; SPI1, isoﬂavone rich soy

protein isolate.

0022-3166/07 $8.00 ª 2007 American Society for Nutrition. 1769

Manuscript received 15 January 2007. Initial review completed 7 March 2007. Revision accepted 11 April 2007.

by guest on November 14, 2012 jn.nutrition.org Downloaded from

C1.html

http://jn.nutrition.org/content/suppl/2007/06/21/137.7.1769.D

Supplemental Material can be found at:carried out in older men for a relatively long duration. None of the

published studies reported equol-excretor status effects on circulating hormone response to soy isoﬂavone interventions in men.

Circulating hormone proﬁles may fail to accurately reﬂect

prostate tissue exposure, and evaluating hormone receptor expression patterns in the prostate may provide additional evidence concerning the role of soy as a cancer preventive dietary

agent. The AR mediates the action of androgens, and AR expression is a potential marker for prostate cancer prognosis (32).

Dietary genistein has been shown to downregulate AR mRNA

expression in rodents (33,34), and genistein has been shown to

suppress AR activity through an estrogen receptor-b (ERb)-

dependent mechanism in LNCaP cells (35). Despite these data,

to our knowledge, there are no studies published to date that

evaluate the effects of soy protein isolate consumption on AR

and ERb expression in men, although one study reported that an

isoﬂavone extract derived from red clover failed to alter AR

expression compared with historically matched controls (26).

The objective of this project was to evaluate the effects of

isoﬂavone-rich soy protein isolate consumption on circulating

concentrations of reproductive hormones and prostate tissue

markers of estrogen and androgen receptor expression in men at

high risk of prostate cancer. The effects of an isoﬂavone-rich soy

protein isolate were compared with those of an isoﬂavone-poor

soy protein isolate to determine whether the isoﬂavones are the

responsible bioactive constituents. The underlying hypothesis

was that isoﬂavone-rich soy protein isolate consumption would

reduce circulating hormones, downregulate AR expression, and

upregulate ERb expression.

Material and Methods

Subjects. Fifty-eight men, aged 50–85 y, were recruited at the

Minneapolis Veteran’s Administration Medical Center Urology Clinic

from a group of patients that had already undergone a transrectal

ultrasound and biopsy. Patients in this study were either at high risk for

developing prostate cancer (n ¼ 53), or had low-grade prostate cancer

that was being followed by active surveillance (n ¼ 5). Subjects were

considered high risk if they had high-grade prostatic intraepithelial neoplasia (PIN) (n ¼ 50) and/or atypical small acinar proliferation (ASAP)

(n ¼ 14). The subjects with prostate cancer had Gleason scores of ,6 and

were not receiving any other prostate cancer therapy. Subjects were

recruited by urologic physicians, and the research nurse reviewed the

patients’ medical records to determine that eligibility criteria were met.

Exclusionary criteria included BMI .40 kg/m

, prostate cancer that required medical treatment, prostatitis, alcohol consumption .14 drinks/

wk, soy or milk allergy, regular antibiotic use, or renal insufﬁciency.

Eighty-seven subjects were screened for the study; 21 chose not to

participate after attending the orientation session, and 66 subjects began

the study. Eight subjects withdrew from the study before their 3-mo

appointment [disliked the study treatment powder (n ¼ 3), inconvenienced by study demands (n ¼ 2), gastrointestinal discomfort (n ¼ 1),

chose conventional prostate cancer treatment (n ¼ 1), weight gain (n ¼

1)]. Three subjects completed 3 mo of the study with good compliance

but chose not to ﬁnish due to inconvenience of the study demands, and

55 subjects completed the full 6-mo study.

Data from 58 subjects were included in the serum hormone analysis,

and 42 subjects were included in the hormone receptor expression

analysis. Fewer participants were eligible for the hormone expression

analysis because 3 subjects did not undergo the ﬁnal prostate biopsy

[liver cancer diagnosis (n ¼ 1), heart condition (n ¼ 1), not clinically

indicated (n ¼ 1)], and 13 subjects had insufﬁcient biopsy tissue at either

baseline or postintervention for the analyses. All 58 subjects who completed the study were Caucasian.

Study design. The University of Minnesota Institutional Review Board

Human Subjects Committee, the Minneapolis Veterans Affairs Institutional Review Board, and the U.S. Army Medical Research and Materiel

Command’s Human Subjects Research Review Board approved the

study protocol, and all subjects provided informed consent, attended an

orientation session, and were provided with a study handbook. During

the study orientation, subjects were interviewed and prompted about

incidental exposure to dietary isoﬂavones (e.g., snack bars, shakes, soy

nuts, canned tuna, legumes, breads) to determine whether they were soy

consumers. Only one participant reported regular soy consumption, but

he did not consume soy-containing products for 1 mo prior to beginning

the study. The 6-mo intervention study used a randomized, singleblinded, placebo-controlled, parallel design. Free-living subjects supplemented their diets with 1 of 3 randomly assigned protein isolates: 1) soy

protein isolate high in isoﬂavones (SPI1); 2) soy protein isolate that had

most of the isoﬂavones removed by alcohol extraction (SPI2); or 3) milk

protein isolate (MPI) (The Solae Company). The protein isolates were

consumed in divided doses twice daily and contributed 40 g/d protein

and 200–400 kcal/d (1 kcal ¼ 4.184 kJ). The isoﬂavone content of the

protein isolates expressed as aglycone equivalents was 107 6 5.0 mg/d

for the SPI1; ,6 6 0.7 mg/d for the SPI2; and 0 mg/d for the MPI

(mean 6 SD). The mean distribution of isoﬂavones was 53% genistein,

35% daidzein, and 11% glycitein in SPI1, and 57% genistein, 20%

daidzein, and 23% glycitein in SPI2 as analyzed by Dr. Pat Murphy

(Department of Food Science and Human Nutrition, Iowa State University). The packets of protein isolate were numbered and patients were

unaware of the treatment protein isolate they had been assigned until all

subjects completed the intervention. Only the study coordinators who

administered the protein isolates knew the group to which each participant belonged. Compliance was assessed by counting the number of

times the patient consumed the protein isolate, as self-reported in recording calendars given to them, and mean compliance was 94%. Dietary and herbal supplements were allowed, and participants were asked

to avoid changing dosages or adding new supplements to their regimen

during the study. Subjects consumed their habitual diets, and received

detailed instructions to exclude soy products to minimize isoﬂavone

consumption from other sources.

Serum collection and analysis. Fasting blood was collected in the

morning at 0, 3, and 6 mo. Serum was separated and aliquots were frozen

at –70 C until analysis. All serum samples were analyzed for testosterone, free testosterone, DHT, androstanediol glucuronide (3a-AG),

androstenedione, dehydroepiandrosterone sulfate (DHEAS), SHBG, estradiol, and estrone. Steroid hormones were analyzed in duplicate by

RIA, and SHBG was analyzed by immunoradiometric assay (Diagnostics

Systems Laboratories). Hormone analyses were performed in 3 batches

and all assays required

125

I-labeled analyte. Intraassay variabilities were

3.7% for testosterone, 4.4% for free testosterone, 6.1% for DHT, 4.5%

for 3a-AG, 4.4% for androstenedione, 2.3% for DHEAS, 4.4% for

SHBG, 3.9% for estradiol, and 4.3% for estrone. An internal control

was utilized to determine variability among batches, and interassay variabilities were between 9 and 30% for all analytes. All 3 serum samples

for each participant were analyzed in the same batch.

Urine collection and analysis. To assess equol-producer status, 24-h

urine was collected in plastic containers containing 1 g/L of ascorbic acid

and separated into aliquots after the addition of sodium azide to a ﬁnal

concentration of 0.1%. Aliquots were frozen at –20 C until analysis.

Equol was determined by HPLC and MS as previously described (36).

The intraassay CV for equol was 8.2%, and the interassay CV was

12.5%. Subjects were classiﬁed as equol excretors if 24-h urine equol

levels exceeded 1000 nmol/d.

Dietary intake and analysis. Food records were completed for 3 d

before each clinic visit. A registered dietitian taught study participants

how to keep accurate food records. Patients were encouraged to use

household scales and volumetric tools and to submit food labels from

unusual foods. Study coordinators reviewed each food record for

completeness and clariﬁed ambiguities with the participant at each clinic

visit. Food records were analyzed with Nutritionist V, version 2.3 (37),

and, for each 3-d food record, mean intakes of energy, macronutrients,

saturated fat, cholesterol, ﬁber, vitamin D, vitamin E, calcium, selenium,

and zinc were calculated.

1770 Hamilton-Reeves et al.

by guest on November 14, 2012 jn.nutrition.org Downloaded from Tissue collection and analysis. Biopsies were performed before the

initial screening and at the 6-mo clinical visit. Biopsy cores were formalinpreserved for 24 h and parafﬁn embedded. The histological diagnoses

were determined during a routine pathological evaluation. Immunohistochemistry was performed to assess AR and ERb expression on primarily normal, hyperplastic, or preneoplastic glands collected from

eligible study participants. Antigen retrieval was achieved by pressure

cooking deparafﬁnized and rehydrated tissue sections at 103 kPa in

citrate buffer. Sections were treated in quenching solution (3% H2O2 in

100% methanol), and then incubated with a protein-blocking solution

(10% milk, 5% serum, and 1% BSA). Samples were incubated overnight

at 4 C with rabbit polyclonal anti-ERb antibody (ab3577; Abcam;

1:1000) for the ERb assay, or for 30 min at room temperature with the

mouse monoclonal anti-AR antibody (AM256–2M; BioGenex; RTU) for

the AR assays. Next, the avidin-biotin peroxidase method was carried

out (Vectastain Elite ABC kit, Vector Laboratories). Color reaction was

developed using diaminobenzidine as the chromagen. Appropriate positive and negative controls were included in all staining runs. Disrupted

glands and glands on the edge of tissue sections were excluded from

analysis to avoid false positives. A technician without prior knowledge of

histological grading scored both the intensity of immunostaining and

the percentage of immunopositive areas at 403 magniﬁcation using

the HSCORE system as previously described (38). The range of the

HSCORE is a minimum of 1 and a maximum of 4 (1 indicated absent

staining; 4 indicated intense staining). A mean of 6 intact glands (range:

2–15) per slide for ERb and a mean of 8 intact glands (range: 3–19) per

slide for AR were averaged to derive the HSCORE (Fig. 1).

Excluded from analysis. The following data were excluded from

statistical analysis: 6-mo dietary intake from one participant reporting

unusually low consumption (mean ,500 kcal) (1 kcal ¼ 4.184 kJ)

during the 3-d food diary as a result of illness; 3 mo DHEAS that was

above normal range (16 mmol/L) and inconsistent with the participant’s

baseline and 6-mo measurements; all DHEAS measurements from one

participant with abnormally high 3-mo and 6-mo DHEAS concentrations (9 and 10 mmol/L, respectively) compared with baseline; and all

SHBG measurements from one subject with undetectable SHBG in the

serum (,3 nmol/L). One subject did not consume the treatment powder

for 3 d prior to his 6-mo appointment as a result of illness, so he was

excluded from the 6-mo equol excretion analysis.

Statistical analysis. The data appeared normally distributed and had

similar variance among groups. Demographic comparisons between

groups were performed with 1-way ANOVA for continuous endpoints,

and chi-square for categories of prostate cancer markers. ANCOVA was

used to compare groups adjusted for the baseline value of the ﬁnal

endpoint. For androstenedione, the model included a treatment by baseline interaction. Preplanned pairwise comparisons of all groups are

reported for each endpoint as dictated by the study hypotheses: each

group’s adjusted mean (least squares mean) was compared with the other

2 groups’ adjusted means. Paired t tests were used to test for signiﬁcant

within-group changes over time. In addition, these covariates were

screened as adjusters: baseline body weight, equol excretor status, and

energy and nutrient intake. P , 0.05 was considered signiﬁcant. All

analyses were performed using SAS, version 9.1 (39).

Results

Baseline. Baseline anthropometrics, cancer status, and dietary

intake did not differ among the groups (Table 1), except that the

MPI group had a higher body weight and the SPI2 group consumed signiﬁcantly more protein, calcium, and zinc at baseline

(Table 2). Baseline prostate steroid receptor expression patterns

(Table 3) and serum hormone and SHBG concentrations (Table

4) did not differ among the groups.

Anthropometrics and dietary intake. Body weight did not

change from baseline to 3 or 6 mo in any group (Table 2), and

the signiﬁcant differences in body weight among the groups at

baseline were maintained. Protein, calcium, and vitamin D intakes increased in all groups during the study as a result of their

concentrations in the protein isolates, and the differences in

protein, calcium, and zinc intake at baseline were not present at

3 and 6 mo. At 3 mo, total and saturated fat consumption were

reduced in the SPI2 group relative to baseline. During the study,

energy, carbohydrate, cholesterol, ﬁber, vitamin E, selenium, and

zinc intakes did not change for any group. Dietary and herbal

supplement usage did not differ among groups (data not shown).

Body weight and protein intake differences among groups were

unrelated to altered hormone concentrations or steroid receptor

expression patterns.

Steroid receptors. Baseline-adjusted AR expression was lower

in prostate biopsies after 6 mo in the SPI1 group compared with

the MPI group (P ¼ 0.04) and tended to be lower in the SPI2

group compared with the MPI group (P ¼ 0.09; Table 3). AR

expression signiﬁcantly increased from baseline in the MPI group,

but not in the other 2 groups. There were no changes from

baseline in ERb expression among the groups (Table 3).

Serum estrogens. The serum estradiol concentration was

signiﬁcantly increased in the SPI2 group at 3 and 6 mo relative

to baseline, and by 6 mo, baseline-adjusted estradiol concentrations were signiﬁcantly higher in the SPI2 group compared with

the other 2 groups (Table 4). Serum estrone was also signiﬁcantly

increased in the SPI2 group at 3 and 6 mo, and was signiﬁcantly

higher than in the MPI group at 3 mo but not at 6 mo.

Serum androgens and SHBG. The serum androstenedione

concentration was signiﬁcantly higher in the SPI1 group than in

the MPI group at 3 mo. At 6 mo it was signiﬁcantly greater than

at baseline in the SPI2 group, resulting in a signiﬁcantly higher

concentration than in the SPI1 group (Table 4). At both 3 and 6

mo, serum DHEAS was higher in the SPI group than in the other

2 groups, and at 3 mo, 3a-AG was higher in the SPI2 group than

the other 2 groups. At 3 mo, the DHT concentration decreased

from baseline in the SPI group. Serum SHBG concentrations

were decreased signiﬁcantly from baseline at 3 and 6 mo in all

groups, with no difference among the groups.

Equol-excretor status and hormone proﬁles. Equol excretor

status was assessed only in the SPI1 group, because only they

consumed sufﬁcient daidzein to excrete equol. At 3 mo, there

were 4 excretors and 15 nonexcretors, but of this group, only

1 excretor remained at 6 mo [dropped out after 3 mo (n ¼ 1),

TABLE 1 Baseline characteristics of subjects

SPI1 SPI2 MPI

n 20 20 18

Age, y 68 6 8 68 6 5 68 6 7

Body wt, kg 91 6 16

88 6 12

98 6 15

Height, cm 175 6 7 173 6 8 176 6 8

BMI, kg/m

30 6 5 29 6 4 32 6 6

Prostate cancer markers,

n (%)

PIN 15 (75) 12 (60) 13 (72)

ASAP 3 (15) 7 (35) 3 (17)

CaP 2 (10) 1 (5) 2 (11)

Values are means 6 SD or n (%). Means in a row with superscripts without a

common letter differ, P , 0.05.

Subjects were categorized by most advanced prostate cancer marker.

Soy effects on hormones in men 1771

by guest on November 14, 2012 jn.nutrition.org Downloaded from apparently changed status (n ¼ 1), and excluded data (n ¼ 1)].

Baseline characteristics (Supplemental Table 1) and serum

hormone concentrations at 3 mo (Supplemental Table 2) did

not differ between excretors and nonexcretors.

Discussion

The present study evaluated men at high risk of prostate cancer

to determine the effects of soy protein consumption on serum

hormones and prostate tissue steroid receptor expression levels.

The major ﬁnding was lower AR expression levels and no differences in ERb expression or circulating hormones in men

consuming SPI1 compared with those consuming MPI.

AR increased signiﬁcantly from baseline in the MPI group,

but did not change from baseline in the soy groups. Because AR

expression is expected to increase in this population (40), we

infer that SPI1 apparently prevented or suppressed a rise in AR

expression. Lower tissue AR expression in the SPI1 group is

consistent with research in which dietary phytoestrogens downregulated AR mRNA expression in adult male rats (33,34,41).

Our data differ, however, from those of Jarred et al. (26), who

reported no differences in AR expression patterns between radical prostatectomy patients treated with isoﬂavones and historically matched controls. The inconsistent results between the

2 studies can be explained by several methodological differences.

In the study by Jarred et al. (26), the subjects, who consumed

160 mg/d of isoﬂavones in extracts derived from red clover, were

men with advanced prostatic neoplasms treated for short and

varied time periods (7–54 d). The tissue sections studied from

the radical prostatectomies taken from treated subjects represented cancerous glandular acinae and were compared with

sections of cancers from historically matched controls. Our

subjects consumed 107 mg/d of isoﬂavones in isoﬂavone-rich

SPI, were earlier in the carcinogenesis continuum, were treated

TABLE 2 Anthropometrics and dietary intake of men at high

risk of prostate cancer that consumed various

protein isolates for 6 mo

SPI1 SPI2 MPI

20 20 18

Weight, kg

Baseline 91 6 16

88 6 12

98 6 15

3 91 6 16

87 6 12

98 6 15

6 90 6 16

87 6 13

99 6 15

Height, cm

Baseline 175 6 16 173 6 8 176 6 8

BMI, kg/m

Baseline 30 6 5 29 6 4 32 6 6

3 30 6 5 29 6 4 32 6 6

6 30 6 5 29 6 4 32 6 6

Energy intake,

kcal/d

Baseline 2140 6 620 2260 6 660 2070 6 520

3 2220 6 720 2030 6 390 2180 6 510

6 2240 6 410 2120 6 670 2330 6 410

Protein, g/d

Baseline 83 6 21

100 6 24

81 6 25

3 Mo *118 6 24 *117 6 16 *121 6 30

6 Mo *118 6 21 *124 6 29 *120 6 18

Carbohydrate, g/d

Baseline 256 6 106 262 6 118 236 6 59

3 Mo 246 6 97 230 6 82 232 6 75

6 Mo 251 6 61 232 6 89 256 6 68

Total fat, g/d

Baseline 86 6 33 93 6 32 88 6 24

3 Mo 80 6 39 *74 6 18 73 6 30

6 Mo 83 6 34 80 6 34 89 6 26

Saturated fat, g/d

Baseline 27 6 11 34 6 14 28 6 11

3 Mo 27 6 13 *26 6 7 24 6 12

6 Mo 26 6 10 29 6 14 30 6 10

Cholesterol, mg/d

Baseline 324 6 202 382 6 153 301 6 163

3 Mo 307 6 131 296 6 115 312 6 233

6 Mo 328 6 147 348 6 175 329 6 234

Fiber, g/d

Baseline 17 6 9 18 6 7 16 6 5

3 Mo 16 6 8 17 6 8 15 6 7

6 Mo 15 6 9 16 6 9 15 6 5

Vitamin D, mg/d

Baseline 4 6 3 4 6 5 4 6 3

3 Mo *9 6 4 *8 6 3 *8 6 2

6 Mo *8 6 2 *8 6 3 *9 6 2

Vitamin E, mg/d

Baseline 8 6 7 8 6 5 6 6 4

3 Mo 6 6 4 7 6 10 6 6 3

6 Mo 7 6 7 6 6 3 6 6 3

Calcium, mg/d

Baseline 890 6 400

1230 6 970

760 6 360

3 Mo *2260 6 440 *2120 6 350 *2200 6 380

6 Mo *2180 6 290 *2340 6 840 *2190 6 340

Selenium, mg/d

Baseline 0.08 6 0.05 0.09 6 0.03 0.08 6 0.05

3 Mo 0.08 6 0.03 *0.06 6 0.03 0.10 6 0.11

6 Mo 0.07 6 0.03 *0.07 6 0.02 0.44 6 1.6

Zinc, mg/d

Baseline 10 6 6

14 6 5

10 6 5

3 Mo 11 6 4 10 6 8 10 6 3

6 Mo 9 6 3 10 6 5 9 6 3

TABLE 2 Continued

All values are means 6 SD. Means in a row with superscripts without a common

letter differ, P , 0.05. *Different from baseline, P , 0.05.

Sample sizes are for all time points except the following: 3 mo, MPI (n ¼ 17), and 6

mo, SPI1 (n ¼ 18), and SPI2 (n ¼ 18).

1 kcal ¼ 4.184 kJ.

FIGURE 1 Representative immunohistochemical staining of AR in

human prostate core biopsies for HSCORE. Arrow indicates stained

acinar cell in MPI control group (enlarged view inset in lower right).

1772 Hamilton-Reeves et al.

by guest on November 14, 2012 jn.nutrition.org Downloaded from for 6 mo each, and all biological samples were evaluated within

the same subject before and after the intervention. Furthermore,

the gland acinae studied presented either benign, hyperplastic, or

preneoplastic tissue.

Consumption of SPI1 did not affect ERb expression or

circulating hormones. The ERb expression results are inconsistent with studies in animals in which prolonged isoﬂavone exposure decreased ERb expression (33,42), and may be explained

by the variability in commercially available ERb antibodies (43).

Our hormone results, however, are consistent with most published reports from the clinical setting. The testosterone results

are consistent with numerous soy or isoﬂavone intervention

studies in which no change in total testosterone was observed

(12–31), but differ from 2 studies of short duration (10,11). Our

ﬁnding of no effect on directly measured free testosterone is

similar to published soy or isoﬂavone intervention studies to

date (11,14,15,20,22,24), and our ﬁnding of no effect on circulating DHT is consistent with most reports (10,14,16,19–

21,23,30), although it differs from results of 2 studies (13,17),

one of which used red clover extract (17). The lack of effect on

circulating estradiol or estrone is consistent with the literature

(10,11,15,16,19,22,29,30), although there is one report of decreased estrone in men consuming soymilk for 8 wk (15).

Serum SHBG decreased signiﬁcantly from baseline in all

study groups. The ﬁnding that consumption of SPI1 decreased

SHBG is similar to a report by Mackey et al. (12); however, they

did not ﬁnd a signiﬁcant decrease in SHBG with an isoﬂavonepoor protein isolate as we did. In contrast to our ﬁndings,

Habito et al. (16) reported increased SHBG in men consuming

35 g of tofu daily for 2 wk, and others have reported no signiﬁcant changes of SHBG with isoﬂavone-rich foods or extracts

(13,15,17,20–23,30). Decreased SHBG is a potentially harmful

effect because SHBG-bound hormones are less biologically available to stimulate hormone-sensitive cancers. Because high protein intake has been associated with decreased SHBG (44), it is

likely that the decrease in SHBG from baseline in all groups in

our study resulted from the subjects’ signiﬁcantly increased

protein intake during the study (45).

The hormonal effects in the SPI2 group were unexpected.

Although AR expression was not signiﬁcantly lower in the SPI2

group, AR expression appeared to be intermediate between that

of SPI1 and MPI groups. In addition, serum estradiol was increased in the SPI2 group. These results are similar to a study in

young men by Dillingham et al. (20) in which a low-isoﬂavone

protein isolate containing ,2 mg/d isoﬂavones signiﬁcantly increased estradiol and estrone compared with a milk protein isolate after a 8-wk intervention. Our results differ, however, from a

study in older men by Goldin et al. (19) in which a low-isoﬂavone

soy protein isolate containing ,2 mg/d isoﬂavones did not change

estradiol or estrone concentrations after a 6-wk intervention.

Interestingly, we found serum estradiol was signiﬁcantly higher in

the SPI2 group than in the SPI1 group, whereas in Dillingham

et al. (20) found that estradiol in the low-isoﬂavone group did not

differ from the high-isoﬂavone group (20).

Serum androstenedione and DHEAS concentrations were

increased in the SPI2 group compared with both SPI1 and MPI

groups. No other soy protein or isoﬂavone intervention study has

TABLE 4 Serum hormones and SHBG in men at high risk

of prostate cancer that consumed various protein

isolates for 6 mo

SPI1 SPI2 MPI

20 20 18

Estradiol, pmol/L

Baseline 67 6 4 66 6 4 69 6 3

3 Mo 75 6 5 *76 6 5 *62 6 6

6 Mo 69 6 3

*79 6 3

66 6 3

Estrone, pmol/L

Baseline 157 6 15 141 6 10 158 6 8

3 Mo 150 6 8

*170 6 8

146 6 8

6 Mo 152 6 10 *171 6 10 150 6 10

Androstenedione, nmol/L

Baseline 2.9 6 0.3 2.9 6 0.3 2.5 6 0.2

3 Mo 3.0 6 0.2

3.0 6 0.2

2.8 6 0.2

6 Mo 2.6 6 0.2

*3.4 6 0.2

2.9 6 0.2

Androstanediol glucuronide, nmol/L

Baseline 19 6 3 18 6 5 16 6 2

3 Mo 17 6 2

24 6 2

17 6 2

6 Mo 16 6 2 20 6 2 18 6 2

DHEAS,

nmol/L

Baseline 2202 6 390 2052 6 300 1977 6 370

3 Mo 2040 6 103

2715 6 103

2126 6 103

6 Mo 1937 6 154

2372 6 146

1946 6 150

DHT, pmol/L

Baseline 1547 6 190 1354 6 170 1072 6 110

3 Mo 1242 6 81 *1076 6 79 1119 6 100

6 Mo 1215 6 94 1174 6 89 1229 6 105

Testosterone, nmol/L

Baseline 12 6 1 13 6 1 12 6 1

3 Mo 13 6 0.5 13 6 0.6 11 6 0.6

6 Mo 13 6 0.6 13 6 0.5 12 6 0.6

Free testosterone, pmol/L

Baseline 33 6 3 34 6 2 29 6 2

3 Mo 33 6 1 33 6 1 32 6 1

6 Mo 32 6 1 32 6 1 31 6 1

SHBG,

nmol/L

Baseline 63 6 7 64 6 8 69 6 9

3 Mo *56 6 3 *56 6 2 *56 6 3

6 Mo *54 6 3 *61 6 3 *58 6 3

Baseline data are unadjusted means 6 SEM. All other data are least-squares means

adjusted for baseline measurement 6 SEM, except androstenedione, which is

additionally adjusted for interaction between treatment and baseline. Means in a row

with superscripts without a common letter differ, P , 0.05. *Different from baseline,

P , 0.05.

Sample sizes are for all time points except: 3 mo MPI (n ¼ 17), and 6 mo SPI1 (n ¼

18), and SPI2 (n ¼ 19).

Sample sizes differed from other hormones due to excluded data. At 3 mo, SPI1

(n ¼ 19) and SPI2 (n ¼ 19). At 6 mo, SPI1 (n ¼ 17) and SPI2 (n ¼ 19).

Sample sizes differed from other hormones due to excluded data. At 3 mo, SPI1

(n ¼ 19), and at 6 mo, SPI1 (n ¼ 18).

TABLE 3 Steroid receptor expression of men at high risk of

prostate cancer that consumed various protein

isolates for 6 mo

SPI1 SPI2 MPI

Androgen receptor HSCORE

n 14 16 14

Baseline 1.37 6 0.06 1.28 6 0.06 1.23 6 0.06

6 Mo 1.26 6 0.05

1.30 6 0.05

*1.42 6 0.05

Estrogen receptor-b

n 14 14 15

Baseline 1.22 6 0.06 1.32 6 0.06 1.23 6 0.06

6 Mo 1.16 6 0.06 1.18 6 0.06 1.26 6 0.05

Baseline data are unadjusted means 6 SEM. All other data are least-squares means

adjusted for baseline measurement 6 SEM. Means in a row with superscripts without

a common letter differ, P , 0.05. *Different from baseline, P , 0.05.

Soy effects on hormones in men 1773

by guest on November 14, 2012 jn.nutrition.org Downloaded from reported a change in circulating androstenedione (12,17,19,20,30),

but all other studies to date have intervened for a shorter duration. Higher DHEAS is consistent with other low-isoﬂavone

soy protein isolate interventions (19,20). Although DHEAS and

androstenedione can be converted by 17b-hydroxysteroid dehydrogenase to testosterone, no signiﬁcant changes were observed

in circulating testosterone, free testosterone, or DHT. Instead,

our study population had low, but normal, testosterone concentrations throughout the study. Although DHEAS and androstenedione concentrations have been associated with aggressive

prostate cancer (46), our ﬁndings of unchanged testosterone and

a trend toward lower AR expression (P ¼ 0.09) suggest neutral effects of SPI2 consumption. In fact, because DHEAS and

androstenedione may be converted to estradiol and estrone in the

prostate gland (47), the increase in DHEAS and androstenedione

may have contributed to the observed increases in circulating

estradiol and estrone. The hormonal effects of SPI2 consumption are likely due to the effects of the alcohol extraction process

on SPI constituents.

In conclusion, we found that consumption of isoﬂavone-rich

soy protein for 6 mo lowered AR expression levels in the prostate,

but did not change ERb expression or circulating hormones in

men at high risk of prostate cancer. Although consumption of

the alcohol-extracted soy protein did not signiﬁcantly lower AR

expression, its effect appeared to be intermediate to that of SPI1

and MPI consumption, suggesting that the isoﬂavones alone may

not be responsible for the AR expression decrease, or, alternatively, that the low level of isoﬂavones in SPI2 were sufﬁcient to

alter the AR. Unexpectedly, consumption of SPI2, but not SPI1,

signiﬁcantly increased estradiol and androstenedione concentrations. None of these results were inﬂuenced by equol excretion

status. These data suggest that consumption of isoﬂavone-rich

and isoﬂavone-poor soy protein isolate exert differing effects on

endogenous hormones and receptor expression, which may

mediate prostate cancer preventive effects.

Acknowledgments

Immunohistochemistry was performed by Kenji Takamura. The

authors thank Kayla Vettling and Ellie Wiener for tissue scoring,

and Lori Sorensen, Nicole Nelson, and Mary McMullen for

assistance with clinic visits and data entry.

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Bilimsel çalışma 3

Isoflavone supplements stimulated the production of serum equol and decreased the serum dihydrotestosterone levels in healthy male volunteers

M Tanaka1, K Fujimoto1, Y Chihara1, K Torimoto1, T Yoneda1, N Tanaka1, A Hirayama1, N Miyanaga2, H Akaza2 and Y Hirao1

1. 1Department of Urology, Nara Medical University, Kashihara, Japan

2. 2Department of Urology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan

Correspondence: Dr M Tanaka, Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. E-mail: masa-t@naramed-u.ac.jp

Received 28 October 2008; Revised 13 March 2009; Accepted 13 March 2009; Published online 14 July 2009.

The aim of this study was to evaluate the effect of supplementing healthy men with soy isoflavones on the serum levels of sex hormones implicated in prostate cancer development. A total of 28 Japanese healthy volunteers (18 equol producers and 10 equol non-producers) between 30 and 59 years of age were given soy isoflavones (60 mg daily) supplements for 3 months, and the changes in their sex hormone levels were investigated at the baseline and after administration. The serum and urine concentrations of daidzein, genistein, and the levels of equol in the fasting blood samples and 24-h stored urine samples were also measured. All 28 volunteers completed the 3-month supplementation with isoflavone. No changes in the serum levels of estradiol and total testosterone were detected after 3-month supplementation. The serum levels of sex hormone-binding globulin significantly increased, and the serum levels of free testosterone and dihydrotestosterone (DHT) decreased significantly after 3-month supplementation. Among the 10 equol non-producers, equol became detectable in the serum of two healthy volunteers after 3-month supplementation. This study revealed that short-term administration of soy isoflavones stimulated the production of serum equol and decreased the serum DHT level in Japanese healthy volunteers. These results suggest the possibility of converting equol non-producers to producers by prolonged and consistent soy isoflavones consumption.

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