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Division of Genetic and Reproductive Toxicology and * R.O.W. Sciences Incorporated, Jefferson Laboratories, National Center for Toxicological Research, Jefferson, AR 72079
2To whom correspondence should be addressed. E-mail: wbranham{at}nctr.fda.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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43 to 0.00008; E2 = 100). These data can be utilized before animal testing to rank order estimates of the potential for in vivo estrogenic activity of a wide range of untested plant chemicals (as well as other chemicals) based on ER binding.
KEY WORDS: • uterus • estrogen receptor • phytoestrogen • mycoestrogen • structure-activity relationship • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
), alteration of uterine (4
–6
) and behavioral (7
,8
) development in rodents, and increases in the volume of the sexually dimorphic nucleus in female rats (9
). More recently, neonatal exposure to genistein has been shown to induce both uterine and vaginal abnormalities similar to those caused by the potent estrogen, ethinyl estradiol (10
). Indeed, some researchers suggest that studies involving laboratory animals ideally should use phytoestrogen-free diets to ensure that no inadvertent estrogen exposure occurs (11
,12
). Despite these adverse effects observed in animals, dietary phytoestrogens might be beneficial in humans (13
–16
).
Although the hazards of human exposure to potent synthetic estrogens (17
,18
) or antiestrogens with estrogen agonist activity (19
–21
) are well documented, the potentially hazardous or beneficial effects of estrogens of plant origin are less well studied. Sonnenschein and Soto (22
) indicate that exogenous estrogens of environmental origin can alter an organism or population by disrupting normally functioning endocrine or reproductive systems by inappropriately mimicking endogenous hormone actions, inhibiting hormone action, modulating hormone production or altering hormone receptor populations.
Under normal conditions, the estrogen receptor (ER)3
is either unliganded or bound by its major endogenous ligand, 17ß-estradiol (E2). This ligand-ER binding results in conformational changes and activation of the ER that allows binding of the ER-ligand complex to nuclear estrogen response elements. This sequence ultimately leads to gene transcription and translation (23
–25
). The ER-ligand competitive binding assay quantitatively assesses a chemical’s ability to bind to the ER. Work in our laboratory (26
) and others (27
) seeks to construct in vitro test systems to assess large numbers of environmental estrogens. The data from these in vitro assays can be used to rank order chemicals that may act via the ER system in vivo in a manner similar to that of the endogenous estrogen (28
).
The purpose of this study was to assess the ER binding of several groups of compounds of plant (flavones, isoflavones, flavanones, coumarins, chalcones) or fungal origin (zearalanone derivatives). These data and others (26
) are being utilized to develop a battery of computational models to predict ER binding of additional plant- and fungus-derived chemicals as well as synthetic environmental chemicals. Quantitative Structure Activity Relationship (QSAR) and other Structure Activity Relationship (SAR) models have shown the utility of this assay approach to provide quantitative information for priority setting for conducting further evaluations of estrogenicity (29
,30
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Adult Sprague-Dawley–derived rats, bred and maintained as an out-bred colony at the National Center for Toxicological Research, were ovariectomized under light ether anesthesia. After an interval of at least 10 d, uteri were removed from groups of 10–12 rats immediately after they were killed by CO2 asphyxiation. To produce a pool of uterine cytosol, the uteri were weighed and homogenized using a Polytron homogenizer at 4°C in TRIS-EDTA-dithiothreitol-glycerol buffer (0.01 L/g tissue) consisting of 10 mmol/L TRIS; 1.5 mmol/L EDTA; glycerol (0.1 L/L buffer); and dithiothreitol (0.154 g/L), pH 7.4. The homogenates were transferred to chilled ultracentrifuge tubes and centrifuged at 105,000 x g for 1 h at 4°C. The supernatants were decanted and frozen at -70°C until used. All procedures involving animals were conducted with strict adherence to guidelines and procedures reviewed and approved by the Institutional Animal Care and Use Committee both within the National Center for Toxicological Research and by an independent review panel.
Assay validation.
All assay procedures outlined here were previously detailed (26
). Uterine cytosol concentration (ER), [3H]-E2, and incubation time and temperature are key parameters for validation of the ER competitive binding assay (27
,31
,32
). Scatchard analyses were used to obtain Kd and Bmax for cytosol concentrations ranging from 10 to 100 g uterine tissue/L of buffer. Bmax was linear with cytosol concentrations in the range of 2–50 g/L (data not shown). At a cytosol concentration of 17 g/L (Bmax = 0.22 nmol/L), a stable Kd of 0.1 nmol/L was observed. This Kd is consistent with current literature values (33
). In addition, at a fixed concentration of 1 nmol/L [3H]-E2, binding increased linearly with increasing receptor concentration. Therefore, the final assay incubation conditions were chosen to be 20 h, 4°C using 17 g/L uterine tissue with 1 nmol/L [3H]-E2.
Assay procedures.
The commercial sources, CAS (Chemical Abstracts Service) designations and chemical purities of the chemicals used are shown in Table 1
. All test compounds were dissolved in 100% ethanol at the highest concentrations achievable, typically 10-2 to 10-3 mol/L. Serial dilutions of the stock solutions in 100% ethanol were made to provide a range of assay concentrations. Each competition assay consisted of 27 tubes; tubes 1 and 26 ("zero competitor" tubes) had no competing E2. Tubes 2 and 27 had a final concentration of 1 x 10-7 mol/L E2, a two orders of magnitude excess of E2 to saturate the ER with nonradiolabeled competitor. The placement of an assay control tube at both the beginning and end of the assay controls for any "tube rack position" effects. The zero competitor tubes containing [3H]-E2 only were assayed to determine total binding, whereas the tubes containing 1 x 10-7 mol/L E2 were assayed to correct for nonspecific binding. Also, in each assay, a complete competition curve for E2 (tubes 2–7 containing a final E2 concentration of 1 x 10-7 mol/L to 3.3 x 10-11 mol/L) was included as an internal assay standard. The test compounds were also dissolved in 100% ethanol, serially diluted in ethanol and assayed at six concentrations for each chemical in assay tubes 8–25. Standards or test compounds (1 x 10-5 L) were combined with 1 x 10-5 L of [3H]-E2 (final concentration of 1 x 10-9 mol/L), 2.3 x 10-4 L of TRIS buffer (50 mmol/L, pH 7.4) and 5 x 10-5 L of stock cytosolic ER in prechilled 12 x 75 borosilicate glass assay tubes. Each assay tube was run in duplicate and each assay was repeated at least once.
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The percentage of binding of the test compounds to the ER was calculated by first subtracting the nonspecific binding (mean dpm in tubes 2 and 27) from the dpm of all of the other tubes. This provides the specifically bound dpm. This number is then expressed as the percentage of [3H]-E2 bound, i.e., specifically bound dpm of the sample/specifically bound dpm of the [3H]-E2 only tubes x 100. The data for each compound are expressed as an IC50, i.e., the molar concentration of the test compound that produces a 50% inhibition of [3H]-E2 binding to the ER. The relative binding activity (RBA) is defined as the ratio of the E2 IC50 to the test compound IC50 multiplied by 100.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-6
; data for the 12 compounds that did not bind to ER or which bound only slightly to ER (5 compounds) are not shown. In many cases, competing ligands were dissolved to the limits of solubility in an attempt to achieve complete binding curves. Nonbinders are defined as chemicals for which an IC50 was not obtained at the highest assay concentration achievable. Several of the phytoestrogens exhibited slight binding at the highest concentrations, but an IC50 concentration was not reached. Most of the curves appeared to be parallel to the E2 standard curve throughout the log-linear portion of the curves. Slight deviations from parallelism were observed with the 3'- and 4'-hydroxyflavanones (Fig. 2
) and the 4- and 4'-hydroxychalcones (Fig. 5
) particularly at concentrations giving >50% competition. These deviations from parallelism did not substantially affect their IC50 values. In all assay replicates, the IC50 values for individual chemicals did not vary by >0.5 log units of concentration. Also, the flavones 6,4'-dihydroxyflavone, 3,6,4'-trihydroxyflavone and apigenin (Fig. 1
), and the chalcones 4- and 4'-hydroxychalcone (Fig. 5
) exhibited an increase in the percentage of [3H]-E2 bound at concentrations that were 
15% of maximum E2 binding. This effect did not alter the IC50 values.
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. Chemical information (CAS number, source, purity) and experimental data derived from the competition curves (IC50, RBA and log RBA) are shown in Table 1
. Of the 46 chemicals tested, 34 exhibited competition for the ER; 3 were strong binders (log RBA >0), 14 were moderate binders (log RBA between 0 and -2), and 17 were weak binders (log RBA -2). Some level of competition for ER binding of [3H]-E2 was seen in some members of all chemical classes; however, the highest affinity phytoestrogen (3,6,4'-trihydroxyflavone) competed with [3H]-E2 binding to the ER with 0.05% lower affinity than E2. Some of the mycoestrogens competed with affinities approaching that of E2. Specific results are described below.
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90% compared with 3,6,4'-trihydroxyflavone. Removal of hydroxyls from the C-ring reduced ER binding (baicalein) or eliminated ER binding (chrysin) as did increasing the number of hydroxyls (fisetin, myricetin, morin, quercetin). Only one methoxyflavone (6-hydroxy-2'-methoxyflavone), which failed to bind ER, was assessed.
Five of the ten flavanones bound to the ER. Single hydroxylation on either the A- or C-rings (6- and 7-hydroxyflavanone and 3'- and 4'-hydroxyflavanone) conferred weak ER binding. This suggests that the phenolic A-ring in steroids is crucial for binding because it can form three hydrogen bonds with ER amino acids and water. Naringenin (the strongest ER-binding flavanone) differs from the flavone apigenin only by the absence of the double bonds at the 2 and 3 carbons of the B-ring. This marginally reduced naringenin ER binding by 75%.
Hydroxylation of the basic isoflavone structure at the 5,7,4' position (genistein) or at the 7,3',4' positions (7,3',4'-trihydroxyisoflavone) resulted in a binding affinity of about 0.45% or 0.00045% of E2, respectively. Conversion of genistein to daidzein by removal of the hydroxyl at the 5 position reduced the affinity for the ER by 95%. Removal of the ketone oxygen at the 4 position and the double bond between C-2 and C-3 in the B-ring (equol) increased ER binding compared with daidzein. Replacement of the 7-hydroxyl of genistein by a methoxy group, thus converting genistein to prunetin, reduced binding to 0.4% of E2.
Chalcone, a nonhydroxylated chemical, exhibited weak binding to the ER, i.e., 0.0015% that of E2. Monohydroxylation of the chalcone structure at the 4- or 4'-carbons bound to ER with 0.003% of the affinity of E2, a slight increase in ER binding. Chalcones with multiple hydroxyls such as 4,2' 4'-trihydroxychalcone and 4,2',4',6'-tetrahydroxychalcone (prunetin) competed with [3H]-E2 for ER with 0.069% and 0.054% lower affinity than E2, respectively.
Only two coumarins [coumestrol and 4-ethyl-7-hydroxy-3-(p-methoxyphenyl)-dihydro-1-benzopyran-2-one] were assessed for ER binding. These each bound to ER with an affinity 0.9% that of E2.
In the group of mycoestrogens selected for assay, the "-anones" differ from the "-enones" by the absence of a double bond between carbons 11 and 12. Zearalanone bound ER with 2% lower affinity than E2. Conversion of the C-7 ketone oxygen to a hydroxyl in the 
stereochemical arrangement increased the ER binding to 43% (-zearalenol) and 30% (-zearalanol) that of E2. Shifting these hydroxyls to the ß arrangement reduced ER binding to about 0.6% that of E2 (ß-zearalanol) and 0.2% that of E2 (ß-zearalenol).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Each assay included a complete competition curve for E2 (positive control) and a negative control (ethanol). The in vitro ER binding assay measures a single bimolecular event, i.e., the reversible binding of a compound to the ER under equilibrium binding conditions established under tightly controlled chemical and physical parameters. This is demonstrated by the simple sigmoid curve on a semilog plot of the reference standard, E2. Two features of the binding curves for several of the phytoestrogens warrant comment. First, four compounds (3'-hydroxyflavanone, 4'-hydroxyflavanone, 4-hydroxychalcone and 4'-hydroxychalcone) yielded binding curves that exhibited an increase in slope in the linear portion of the curve below 50% binding compared with both the E2 standard and their chemical class counterparts. Whether or not this represents evidence of a multistep ER binding mechanism is not known. However, there was little or no effect on the IC50.
Second, several of the flavones (6,4'-dihydroxyflavone, 3,6,4'-trihydroxyflavone and apigenin) and chalcones (4-hydroxychalcone and 4'-hydroxychalcone) exhibited biphasic curves in which there was an increase in the percentage of [3H]-E2 bound at concentrations above 85% of maximal competition. This biphasic curve suggests that very high concentrations of some competitors caused more [3H]-E2 to bind to the ER. Borgna and Ladrech (34
) showed that the dissociation rate of the ER-[3H]-E2 complexes increases at high concentrations of competitor. Although this would establish a different equilibrium for [3H]-E2, this should result in a lowering of apparent [3H]-E2 binding instead of an increase. It has been suggested (personal communication, Dr. V. J. Kramer, Rohm and Haas, Spring House, PA) that the higher apparent ER binding could be an artifact caused by precipitation of these chemicals at high concentrations, carrying [3H]-E2 into the HAP pellet. However, the HAP pellet was washed 4 times, which should dilute the high competitor concentrations. Also, because the ER is present in the assay as a high speed supernatant of a cytosolic extract, the high competitor concentrations may cause [3H]-E2 to bind to other cytosolic proteins, resulting in greater nonspecific binding. Clearly, this phenomenon is not understood.
Recent crystal structure analysis of four ligand-receptor complexes has indicated that the phenolic ring, normally the A-ring in steroids or phytoestrogens, is crucial for binding because it forms three hydrogen bonds with ER amino acids and water (35
–37
). However, a recent crystal structure for ER-ß and genistein showed that the phenolic C-ring served to form the hydrogen bonds (37
). This suggests that a chemical may bind in two orientations that differ by 180°. Our data showing that 3'-hydroxyflavanone and 4'-hydroxyflavanone have a phenolic C-ring important in binding suggests the possibility that "flipping" of these chemicals may occur in a manner similar to the situation for genistein binding to ER-ß.
Although there are no comprehensive analyses of phytoestrogen or mycoestrogen binding to rat ER, our data are consistent with values for several of the more commonly studied compounds. Verdeal et al. (38
), using rat uterine cytosol incubated for 2 h at 20°C and dextran-coated charcoal to remove unbound components, found the following relative binding affinities: E2, 100; coumestrol, 4.9; genistein, 1.3; daidzein, 0.09; biochanin A, 0.07; and zearalenone, 4.0. Similarly, Wang et al. (39
) reported an RBA of 0.9 for genistein and no ER binding for quercetin. Our data are generally within 20% of these values. Gutendorf and Westendorf (40
) reported relative binding affinities for genistein (0.01) and coumestrol (0.12) that are lower than the results reported here and those cited above. Comparison of their ER binding data for "nonphytoestrogen" compounds with our previously published data (26
) show their RBA to be consistently lower. Although these differences are small in some cases, we cannot explain them.
Mycoestrogens have been shown to be relatively potent inducers of uterine weight in rodents (41
). Zearalanol, marketed as Ralgro (zeranol), has long been used as a growth promoter in cattle (42
). The mycoestrogens examined here exhibited the greatest binding to ER of all chemical classes examined. The affinity of -zearalanol to ER is 30% lower than that for E2, which is in agreement with previous assays (43
,44
). Using the yeast cell estrogen screening assay, Coldham et al. (45
) showed that -zearalanol induces an estrogen-responsive reporter gene at a level that is only 1.3% of that induced by E2.
Measurement of estrogen binding to ER correlates with estrogenicity (46
). For chemicals that do not show good correspondence for these two measures, differences in absorption, distribution, metabolism and elimination as well as ligand-specific differences in gene expression may provide an explanation. The estrogenicity of many of the more common phytoestrogens has been established by several measures. Using an in vitro rat endometrium–derived adenocarcinoma cell line, Hopert et al. (47
) showed that coumestrol, genistein and daidzein induce production of the estrogen-regulated uterine protein, complement C3 (48
,49
). Similarly, genistein, biochanin A, daidzein, apigenin, kaempferol and coumestrol have been shown to increase DNA synthesis in MCF-7 cells (50
,51
). Our analyses indicate that these phytoestrogens are generally the more active ER binders. Although the estrogenic activity of phytoestrogens in humans is still under investigation, daily consumption of soy by postmenopausal women significantly increases the serum levels of several phytoestrogens (52
). The serum levels of genistin, daidzin and equol increased by 756, 593 and 1008%, respectively.
Although other publications report ER RBA for structurally diverse data sets (27
,53
–56
), these data, combined with another data set from our laboratory (26
), represent the most comprehensive published data set for naturally occurring chemicals competing with [3H]-E2 for ER assessed under conditions of a single, rigorously validated assay. In addition, these data are sufficiently complete, comprising both ER binders and ER nonbinders, to allow determination of the effects of substituent groups on binding. As such, these data are ideal for input into various computer-based models for QSAR and SAR modeling (29
,30
). We are currently assessing binding of these dietary estrogens to both rat alphafetoprotein (in amniotic fluid) and human sex hormone binding globulin (in plasma from pregnant women). Together, these data sets, in the appropriate computer-based models, will be useful to predict the risk of estrogen exposure.
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FOOTNOTES |
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3 Abbreviations used: CAS, Chemical Abstracts Service; E2, 17ß-estradiol; ER, estrogen receptor; HAP, hydroxylapatite; IC50, the molar concentration of test compound that inhibits E2 binding by 50%; RBA, relative binding activity; SAR, Structure-Activity Relationship; QSAR, Quantitative Structure-Activity Relationship.
Manuscript received 26 August 2001. Initial review completed 15 October 2001. Revision accepted 23 January 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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