***Biochemical and Molecular Action of Nutrients***

**Phytoestrogens and Mycoestrogens Bind to the Rat Uterine Estrogen
Receptor^1^**

William S. Branham,^2^ Stacey L. Dial, Carrie L. Moland, Bruce S. Hass,
Robert M. Blair, Hong Fang,\* Leming Shi,\* Weida Tong,\* Roger G.
Perkins\* and Daniel M. Sheehan

*Division of Genetic and Reproductive Toxicology and \*R.O.W. Sciences
Incorporated, Jefferson Laboratories, National Center for Toxicological
Research, Jefferson, AR 72079*

ABSTRACT Consumption of phytoestrogens and mycoestrogens in food
products or as dietary supplements is of interest because of both the
potential beneficial and adverse effects of these compounds in
estrogen-responsive target tissues. Although the hazards of exposure to
potent estrogens such as diethylstilbestrol in developing male and
female reproductive tracts are well characterized, less is known about
the effects of weaker estrogens including phytoestrogens. With some
exceptions, ligand binding to the estrogen receptor (ER) predicts
uterotrophic activity. Using a well-established and rigorously validated
ER-ligand binding assay, we assessed the relative binding affinity (RBA)
for 46 chemicals from several chemical structure classes of potential
phytoestrogens and mycoestrogens. Although none of the test compounds
bound to ER with the affinity of the standard, 17\~-estradiol (E~2~), ER
binding was found among all classes of chemical structures (flavones,
isoflavones, flavanones, coumarins, chalcones and mycoestrogens).
Estrogen receptor relative binding affinities were distributed across a
wide range (from \~43 to 0.00008; E~2~ \~ 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. J. Nutr.
132: 658--664, 2002.

KEY WORDS: \~ *uterus *\~ *estrogen receptor *\~ *phytoestrogen *\~
*mycoestrogen *\~ *structure-activity relationship *\~ *rats*

Phytoestrogens contained in the diet and in food supple­ments have the
potential for both risks and benefits with respect to human health.
Estrogenic chemicals consumed as constituents of plants (phytoestrogens)
or fungi (mycoestro­gens) exert a variety of adverse effects in animals
including reduction in fertility in sheep grazing on
phytoestrogen-con­taining clover (1--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

1 Supported in part by the FDA's Office of Women's Health, the Chemical
Manufacturer's Association and by the U.S. Department of Energy Oak
Ridge Institute for Science and Education.

2 To whom correspondence should be addressed.

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, 17\~-estradiol
(E~2~). 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).

3 Abbreviations used: CAS, Chemical Abstracts Service;
E~2~,~\ ~17\~-estradiol; ER, estrogen receptor; HAP, hydroxylapatite;
~IC50,\ ~the molar concentration of test compound that inhibits
E~2\ ~binding by 50%; RBA, relative binding activity; SAR,
Structure-Activity Relationship; QSAR, Quantitative Structure-Activity
Relation­ship.

0022-3166/02 \$3.00 © 2002 American Society for Nutritional Sciences.

Manuscript received 26 August 2001. Initial review completed 15 October
2001. Revision accepted 23 January 2002.

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).

**MATERIALS AND METHODS**

***Animals. ***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 CO~2~ asphyxiation. To produce a pool of
uterine cytosol, the uteri were weighed and homogenized using a Polytron
homogenizer at 4°C in TRIS-EDTA-dithiothreitol­glycerol 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
\~ *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),
\[^3^H\]-E~2~, 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 *K*~d\ ~and *~B~*~max\ ~for cytosol
concentrations ranging from 10 to 100 g uterine tissue/L of buffer.
*~B~*~max\ ~was linear with cytosol concentrations in the range of 2--50
g/L (data not shown). At a cytosol concentration of 17 g/L (*^B^*max \~
0.22 nmol/L), a stable *K*d* *of \~0.1 nmol/L was observed. This
*K*~d\ ~is consistent with current liter­ature values (33 ). In addition,
at a fixed concentration of 1 nmol/L \[^3^H\]-E~2~, 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 \[^3^H\]-E~2~.

***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 E~2~. Tubes 2 and 27 had a final concentration of 1 \~ 10\~^7^
mol/L E~2~, a two orders of magnitude excess of E~2~ 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 \[^3^H\]-E~2~
only were assayed to determine total binding, whereas the tubes
containing 1 \~ 10\~^7^ mol/L E~2~ were assayed to correct for
nonspecific binding. Also, in each assay, a complete competition curve
for E~2~ (tubes 2--7 containing a final E~2~ concentration of 1 \~
10\~^7^ mol/L to 3.3 \~ 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 \~ 10\~^5^
L) were combined with 1 \~ 10\~^5^ L of \[^3^H\]-E~2~ (final
concentration of 1 x 10\~^9^ mol/L), 2.3 \~ 10\~^4^ L of TRIS buffer (50
mmol/L, pH 7.4) and 5 \~ 10\~^5^ L of stock cytosolic ER in prechilled
12 \~ 75 borosilicate glass assay tubes. Each assay tube was run in
duplicate and each assay was repeated at least once.

After incubation at 4°C for 20 h, 7.5 \~ 10\~^4^ L of a
hydroxyapatite(HAP) slurry (60% HAP in 50 mmol/L TRIS, pH 7.4) was added
to the reaction mixture tubes. The tubes were vortexed briefly at 5-min
intervals for 20 min and centrifuged for 4 min at 600 \~ *g. *The
supernatant was discarded and the pellet was resuspended in 2 mL of cold
50 mmol/L TRIS buffer, and vortexed as above. This wash was repeated two
times. After the third wash, the pellet was resuspended in 0.002 L of
cold 100% ethanol and vortexed every 5 min for 15 min. After
centrifugation at 600 \~ *g, *the ethanolic supernatant was decanted
directly into scintillation counting vials for determination of
\[^3^H\]-E~2~ remaining in the assay tube.

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 \[^3^H\]-E~2~ bound, i.e., specifically bound dpm of the
sample/specifically bound dpm of the \[^3^H\]-E~2~ only tubes \~ 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
\[^3^H\]-E~2~ binding to the ER. The relative binding activity (RBA) is
defined as the ratio of the E~2~ ~IC50\ ~to the test compound
^IC^50^\ ^multiplied by 100.

**RESULTS**

Binding curves for the 29 compounds that competed with E~2~ for ER
binding are shown in **Figures 1**-**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 E~2~ 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
^IC^50^\ ^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 \[^3^H\]-E~2~ bound at concentrations that were \~15% of maximum E~2~
binding. This effect did not alter the ~IC50\ ~values.

The basic structures and ring designations for the fla­vanoids,
chalcones, coumarins and mycoestrogens are shown in **Figure 7.
**Chemical information (CAS number, source, purity) and experimental
data derived from the competition curves (IC~50~, 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 \[^3^H\]-E~2~ was
seen in some members of all chemical classes; however, the highest
affinity phytoestrogen (3,6,4\~-trihy­droxyflavone) competed with
\[^3^H\]-E~2~ binding to the ER with \~0.05% lower affinity than E~2~.
Some of the mycoestrogens competed with affinities approaching that of
E~2~. Specific re­sults are described below.

All of the flavones that bound ER are hydroxylated at the 4\~ carbon
with the exception of quercetin, a nonbinder. All other nonbinders
lacked 4\~ hydroxyls. Flavones with hydroxyls at the 6,4\~ or 3,6,4\~
carbons exhibited the strongest ER bind­ing. Although neither the parent
flavone structure nor 7-hy-

+---------+---------+---------+---------+---------+---------+--------+
| **660** | BRANHAM |         |         |         |         |        |
|         | ET AL.  |         |         |         |         |        |
|         |         |         |         |         |         |        |
|         | **TABLE |         |         |         |         |        |
|         | 1**     |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| *C      |         |         |         |         |         |        |
| hemical |         |         |         |         |         |        |
| info    |         |         |         |         |         |        |
| rmation |         |         |         |         |         |        |
| and     |         |         |         |         |         |        |
| comp    |         |         |         |         |         |        |
| arisons |         |         |         |         |         |        |
| of rat  |         |         |         |         |         |        |
| uterine |         |         |         |         |         |        |
| cy      |         |         |         |         |         |        |
| tosolic |         |         |         |         |         |        |
| e       |         |         |         |         |         |        |
| strogen |         |         |         |         |         |        |
| r       |         |         |         |         |         |        |
| eceptor |         |         |         |         |         |        |
| (ER)    |         |         |         |         |         |        |
| binding |         |         |         |         |         |        |
| data*   |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| C       | CAS \#  | Sourc   | Pu      | Log RBA |         |        |
| ompound |         | e^1--8^ | rity^9^ |         |         |        |
| Name    |         |         | Mean^   |         |         |        |
|         |         |         | 10^IC50 |         |         |        |
|         |         |         | M       |         |         |        |
|         |         |         | ean^10^ |         |         |        |
|         |         |         | RBA     |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 17\~-Es | 50-28-2 | U.S.    | NA^11^  | 2.00    |         |        |
| tradiol |         | Biochem | 9.0 \~  |         |         |        |
| (st     |         |         | 1       |         |         |        |
| andard) |         |         | 0\~^10^ |         |         |        |
|         |         |         | 1.0 \~  |         |         |        |
|         |         |         | 10^2^   |         |         |        |
|         |         |         |         |         |         |        |
|         |         |         | *Fl     |         |         |        |
|         |         |         | avones* |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 3,6,4\~ |         | I       | Pure    | \~0.35  |         |        |
| -Trihyd |         | ndofine | 2.0 \~  |         |         |        |
| roxy    |         |         | 10\~^7^ |         |         |        |
| flavone |         |         | 4.5 \~  |         |         |        |
|         |         |         | 10\~^1^ |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 6       |         | I       | Pure    | \~0.82  |         |        |
| ,4\~-Di |         | ndofine | 5.9 \~  |         |         |        |
| hydro   |         |         | 10\~^7^ |         |         |        |
| xyflavo |         |         | 1.5 \~  |         |         |        |
| ne      |         |         | 10\~^1^ |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 7,4\    | 5       | Sigma   | 95% 3.2 | \~1.55  |         |        |
| ~,5-Tri | 20-36-5 |         | \~      |         |         |        |
| hydroxy |         |         | 10\~^6^ |         |         |        |
| flavone |         |         | 2.8 \~  |         |         |        |
| (Ap     |         |         | 10\~^2^ |         |         |        |
| igenin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 3       | 5       | Fluka   | \~96%   | \~1.61  |         |        |
| ,5,7,4\ | 20-18-3 |         | 3.7 \~  |         |         |        |
| ~-Tetra |         |         | 10\~6   |         |         |        |
| hydroxy |         |         | 2.5 \~  |         |         |        |
| flavone |         |         | 10\~^2^ |         |         |        |
| (Kaem   |         |         |         |         |         |        |
| pferol) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 3,3     | 5       | Aldrich | NA^11^  | \~2.35  |         |        |
| \~,4\~, | 28-48-3 |         | 2.0 \~  |         |         |        |
| 7-Tetra |         |         | 10\~^5^ |         |         |        |
| hydroxy |         |         | 4.5 \~  |         |         |        |
| flavone |         |         | 10\~^3^ |         |         |        |
| (F      |         |         |         |         |         |        |
| isetin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 3       | 5       | I       | Pure    | \~2.75  |         |        |
| ,5,7,3\ | 29-44-2 | ndofine | 5.1 \~  |         |         |        |
| ~,4\~,5 |         |         | 10\~^5^ |         |         |        |
| \~-Hexa |         |         | 1.8 \~  |         |         |        |
| hydroxy |         |         | 10\~^6^ |         |         |        |
| flavone |         |         |         |         |         |        |
| (Myr    |         |         |         |         |         |        |
| icetin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 5,      | 4       | Aldrich | NA 1.0  | \~3.05  |         |        |
| 6,7-Tri | 91-67-8 |         | \~      |         |         |        |
| hydroxy |         |         | 10\~^4^ |         |         |        |
| flavone |         |         | 9.0 \~  |         |         |        |
| (Bai    |         |         | 10\~^4^ |         |         |        |
| calein) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 3,5,7   | 4       | Sigma   | NA 2.0  | \~3.35  |         |        |
| ,4\~,6\ | 80-16-0 |         | \~      |         |         |        |
| ~-Penta |         |         | 10\~^4^ |         |         |        |
| hydroxy |         |         | 4.5 \~  |         |         |        |
| flavone |         |         | 10\~^4^ |         |         |        |
| Di      |         |         |         |         |         |        |
| hydrate |         |         |         |         |         |        |
| (Morin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| Rutin   | 1       | Sigma   | 95% 1.1 | \~4.09  |         |        |
|         | 53-18-4 |         | \~      |         |         |        |
|         |         |         | 10\~3   |         |         |        |
|         |         |         | 8.2 \~  |         |         |        |
|         |         |         | 10\~5   |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 6-      | 66      | I       | Pure    | ---     |         |        |
| Hydroxy | 65-83-4 | ndofine | NB^11^  |         |         |        |
| flavone |         |         | ---     |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 6-Hyd   |         | I       | NA NB   | ---     |         |        |
| ro      |         | ndofine | ---     |         |         |        |
| xy-2\~- |         |         |         |         |         |        |
| methoxy |         |         |         |         |         |        |
| flavone |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 5,7-Di  | 4       | Sigma   | NA NB   | ---     |         |        |
| hydroxy | 80-40-0 |         | ---     |         |         |        |
| flavone |         |         |         |         |         |        |
| (C      |         |         |         |         |         |        |
| hrysin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 3,5,7   | 61      | Sigma   | NA NB   | ---     |         |        |
| ,3\~,4\ | 51-25-3 |         | ---     |         |         |        |
| ~-Penta |         |         |         |         |         |        |
| hydroxy |         |         |         |         |         |        |
| flavone |         |         |         |         |         |        |
| di      |         |         |         |         |         |        |
| hydrate |         |         |         |         |         |        |
| (Que    |         |         |         |         |         |        |
| rcetin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| Flavone | 5       | Sigma   | NA NB   | ---     |         |        |
|         | 25-82-6 |         | ---     |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 7-      | 66      | I       | Pure NB | ---     |         |        |
| Hydroxy | 65-86-7 | ndofine | ---     |         |         |        |
| flavone |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         | *Falv   |         |         |        |
|         |         |         | anones* |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 5,7,4\  | 936     | Sigma   | 95% 1.2 | \~2.13  |         |        |
| ~-Trihy | 02-28-9 |         | \~      |         |         |        |
| droxyfl |         |         | 10\~^5^ |         |         |        |
| avanone |         |         | 7.5 \~  |         |         |        |
| (\~     |         |         | 10\~^3^ |         |         |        |
| Nari    |         |         |         |         |         |        |
| ngenin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 4\~-Hy  | 65      | I       | Pure    | \~2.65  |         |        |
| droxyfl | 15-37-3 | ndofine | 4.0 \~  |         |         |        |
| avanone |         |         | 10\~^5^ |         |         |        |
|         |         |         | 2.3 \~  |         |         |        |
|         |         |         | 10\~^3^ |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 3\~-Hy  |         | I       | Pure    | \~2.78  |         |        |
| droxyfl |         | ndofine | 5.4 \~  |         |         |        |
| avanone |         |         | 10\~5   |         |         |        |
|         |         |         | 1.7 \~  |         |         |        |
|         |         |         | 10\~3   |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 6       | 42      | I       | Pure    | ---     |         |        |
| Hy      | 50-77-5 | ndofine | SB^11^  |         |         |        |
| droxyfl |         |         | ---     |         |         |        |
| avanone |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 7       | 65      | I       | Pure SB | ---     |         |        |
| Hy      | 15-36-2 | ndofine | ---     |         |         |        |
| droxyfl |         |         |         |         |         |        |
| avanone |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| Fl      | 4       | Aldrich | 95% NB  | ---     |         |        |
| avanone | 87-26-3 |         | ---     |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| \~      | 72      | Sigma   | NA NB   | ---     |         |        |
| C       | 95-85-4 |         | ---     |         |         |        |
| atechin |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 5,7,    | 5       | Sigma   | NA NB   | ---     |         |        |
| 3\~-Tri | 20-33-2 |         | ---     |         |         |        |
| hydroxy |         |         |         |         |         |        |
| -4\~-me |         |         |         |         |         |        |
| thoxyfl |         |         |         |         |         |        |
| avanone |         |         |         |         |         |        |
| (Hesp   |         |         |         |         |         |        |
| eretin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| N       | 102     | Sigma   | \~95%   | ---     |         |        |
| aringen | 36-47-2 |         | NB ---  |         |         |        |
| in-7-ne |         |         |         |         |         |        |
| ohesper |         |         |         |         |         |        |
| idoside |         |         |         |         |         |        |
| (Na     |         |         |         |         |         |        |
| ringin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 3,5,7,3 | 4       | Sigma   | \~98%   | ---     |         |        |
| \~,4\~- | 80-18-2 |         | NB ---  |         |         |        |
| Pentahy |         |         |         |         |         |        |
| droxyfl |         |         |         |         |         |        |
| avanone |         |         |         |         |         |        |
| (Tax    |         |         |         |         |         |        |
| ifolin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         | *Isofl  |         |         |        |
|         |         |         | avones* |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 5,7,4\~ | 4       | Toronto | \~99%   | \~0.35  |         |        |
| -Trihyd | 46-72-0 |         | 2.0 \~  |         |         |        |
| roxyiso |         |         | 10\~^7^ |         |         |        |
| flavone |         |         | 4.5 \~  |         |         |        |
| (Gen    |         |         | 10\~^1^ |         |         |        |
| istein) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 7,4\~   | 5       | S       | NA 6.0  | \~0.82  |         |        |
| -Isofla | 31-95-3 | pectrum | \~      |         |         |        |
| vandiol |         |         | 10\~^7^ |         |         |        |
| (Equol) |         |         | 1.5 \~  |         |         |        |
|         |         |         | 10\~^1^ |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 4\~,    | 4       | Sigma   | \~98%   | \~1.65  |         |        |
| 7-Dihyd | 86-66-8 |         | 4.0 \~  |         |         |        |
| roxyiso |         |         | 10\~^6^ |         |         |        |
| flavone |         |         | 2.3 \~  |         |         |        |
| (Da     |         |         | 10\~^2^ |         |         |        |
| idzein) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 7,      | 4       | I       | Pure    | \~2.35  |         |        |
| 3\~,4\~ | 85-63-2 | ndofine | 2.0 \~  |         |         |        |
| -Trihyd |         |         | 10\~5   |         |         |        |
| roxyiso |         |         | 4.5 \~  |         |         |        |
| flavone |         |         | 10\~3   |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 5,7-D   | 4       | Sigma   | NA 2.1  | \~2.37  |         |        |
| ihydrox | 91-80-5 |         | \~      |         |         |        |
| y-4-met |         |         | 10\~^5^ |         |         |        |
| hoxyiso |         |         | 4.3 \~  |         |         |        |
| flavone |         |         | 10\~^3^ |         |         |        |
| (Bi     |         |         |         |         |         |        |
| ochanin |         |         |         |         |         |        |
| A)      |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 5,4\~-d | 5       | I       | Pure SB | ---     |         |        |
| ihydrox | 52-59-0 | ndofine | ---     |         |         |        |
| y-7-met |         |         |         |         |         |        |
| hoxyiso |         |         |         |         |         |        |
| flavone |         |         |         |         |         |        |
| (Pr     |         |         |         |         |         |        |
| unetin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 7-h     | 4       | I       | Pure SB | ---     |         |        |
| ydroxy- | 85-72-3 | ndofine | ---     |         |         |        |
| 4\~-met |         |         |         |         |         |        |
| hoxyiso |         |         |         |         |         |        |
| flavone |         |         |         |         |         |        |
| (Formon |         |         |         |         |         |        |
| onetin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 6,7,4\~ | 178     | I       | Pure SB | ---     |         |        |
| -Trihyd | 17-31-1 | ndofine | ---     |         |         |        |
| roxyiso |         |         |         |         |         |        |
| flavone |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 4\~,5,7 | 5       | Sigma   | \~95%   | ---     |         |        |
| -Trihyd | 29-59-9 |         | NB ---  |         |         |        |
| roxyiso |         |         |         |         |         |        |
| flavone |         |         |         |         |         |        |
| 7-gl    |         |         |         |         |         |        |
| ucoside |         |         |         |         |         |        |
| (Ge     |         |         |         |         |         |        |
| nistin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         | *Cha    |         |         |        |
|         |         |         | lcones* |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 4,2\~,  | 60-82-2 | Sigma   | NA 1.3  | 6.9 \~  | \~1.16  |        |
| 4\~,6\~ |         |         | \~      | 10\~^2^ |         |        |
| -Tetrah |         |         | 10\~6   |         |         |        |
| ydroxyc |         |         |         |         |         |        |
| halcone |         |         |         |         |         |        |
| (Phl    |         |         |         |         |         |        |
| oretin) |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 4,      | 9       | I       | Pure    | 5.4 \~  | \~1.26  |        |
| 2\~,4\~ | 61-29-5 | ndofine | 1.6 \~  | 10\~^2^ |         |        |
| -Trihyd |         |         | 10\~^6^ |         |         |        |
| roxyc   |         |         |         |         |         |        |
| halcone |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 4\~-H   | 26      | I       | 97% 2.4 | 3.7 \~  | \~2.43  |        |
| ydroxyc | 57-25-2 | ndofine | \~      | 10\~3   |         |        |
| halcone |         |         | 10\~5   |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| 4-H     | 204     | I       | 97% 3.2 | 2.8 \~  | \~2.55  |        |
| ydroxyc | 26-12-4 | ndofine | \~      | 10\~^3^ |         |        |
| halcone |         |         | 10\~^5^ |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| C       | 94-41-7 | I       | 97% 6.0 | 1.5 \~  | \~2.82  |        |
| halcone |         | ndofine | \~      | 10\~^3^ |         |        |
|         |         |         | 10\~^5^ |         |         |        |
|         |         |         |         |         |         |        |
|         |         |         | *Cou    |         |         |        |
|         |         |         | marins* |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| Cou     | 4       | S       | NA 1.1  | 8.2 \~  | \~0.05  |        |
| mestrol | 79-13-0 | pectrum | \~      | 10\~^1^ |         |        |
|         |         |         | 10\~^7^ |         |         |        |
| 4-Et    |         |         |         |         |         |        |
| hyl-7-h |         |         |         |         |         |        |
| ydry-3- |         |         |         |         |         |        |
| (p-meth |         |         |         |         |         |        |
| oxyphen |         |         |         |         |         |        |
| yl)-dih |         |         |         |         |         |        |
| ydro-1‑ |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| be      | 52      | NCI     | NA 1.1  | 8.2 \~  | \~0.05  |        |
| nzopyra | 19-17-0 |         | \~      | 10\~^1^ |         |        |
| n-2-one |         |         | 10\~^7^ |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         | *       |         |         |        |
|         |         |         | Mycoest |         |         |        |
|         |         |         | rogens* |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| \~-Zea  | 364     | Sigma   | NA 2.9  | 10\~^9^ | 4.3 \~  | 1.63   |
| ralenol | 55-72-8 |         | \~      |         | 10^1^   |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| \~-Zea  | 265     | Sigma   | NA 3.0  | 10\~^9^ | 3.0 \~  | 1.48   |
| ralanol | 38-44-3 |         | \~      |         | 10^1^   |        |
+---------+---------+---------+---------+---------+---------+--------+
| Zear    | 59      | Sigma   | NA 4.3  | 10\~8   | 2.1 \~  | 0.32   |
| alanone | 75-78-0 |         | \~      |         | 100     |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| \~-Zea  | 424     | Sigma   | 98% 1.4 | 10\~^7^ | 6.4 \~  | \~0.19 |
| ralanol | 22-68-4 |         | \~      |         | 10\~^1^ |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+
| \~-Zea  | 710     | Sigma   | NA 4.4  | 10\~^7^ | 2.0 \~  | \~0.69 |
| ralenol | 30-11-0 |         | \~      |         | 10\~^1^ |        |
+---------+---------+---------+---------+---------+---------+--------+
|         |         |         |         |         |         |        |
+---------+---------+---------+---------+---------+---------+--------+

1 Aldrich, Milwaukee, WI.

2 Fluka Chemical, Milwaukee, WI.

3 Indofine Chemical, Somerville, NJ.

4 Sigma, St. Louis, MO.

5 Spectrum Chemical, Gardena, CA.

6 Toronto Research Chemicals, North York, Ontario, Canada.

7 United States Biochemical, Cleveland, OH.

8 Drug Synthesis and Chemistry Branch, Developmental Therapeutics
Program, Division of Cancer Treatment, National Cancer Institute,
Bethesda, MD.

9 Purity information as provided by the vendor/manufacturer.

10 The data represent the means of at least two independent binding
assays.

11 CAS, Chemical Abstracts Service; RBA, relative binding affinity; NA,
not available; NB, nonbinder; SB, slight binder.

**FIGURE 1 **Competition curves for flavone binding to rat uterine
cytosolic estrogen receptor (ER). In this assay, \[^3^H\]-17\~-estradiol
(E~2~) is competing for binding to the ER with increasing concentrations
of either nonradiolabeled E~2~ (assay standard) or the flavones. Each
datum rep­resents the mean of at least 2 independent binding assays. The
com­petitor concentration at 50% reduction in \[^3^H\]-E~2~ binding
(IC~50~) is found at the intersection of the binding curves with the 50%
binding line ( ).

droxyflavone bound to ER, hydroxylation of carbon-6 con­ferred weak
binding. Placement of hydroxyls at the 5 and 7 positions (apigenin) or
an additional hydroxyl at the 3 posi­tion (kaempferol) reduced ER binding
by \~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.

**FIGURE 3 **Competition curves for isoflavone binding to rat uterine
cytosolic estrogen receptor (ER). In this assay, \[^3^H\]-17\~-estradiol
(E~2~) is competing for binding to the ER with increasing concentrations
of either nonradiolabeled E~2~ (assay standard) or the isoflavones. Each
datum represents the mean of at least 2 independent binding assays. The
competitor concentration at 50% reduction in \[^3^H\]-E~2~ binding
(IC~50~) is found at the intersection of the binding curves with the 50%
binding line ( ).

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%.

**FIGURE 2 **Competition curves for flavanone binding to rat uterine
cytosolic estrogen receptor (ER). In this assay, \[^3^H\]-17\~-estradiol
(E~2~) is competing for binding to the ER with increasing concentrations
of either nonradiolabeled E~2~ (assay standard) or the flavanones. Each
datum represents the mean of at least 2 independent binding assays. The
competitor concentration at 50% reduction in \[^3^H\]-E~2~ binding
(IC~50~) is found at the intersection of the binding curves with the 50%
binding line ( ).

**FIGURE 4 **Competition curves for coumarin binding to rat uterine
cytosolic estrogen receptor (ER). In this assay, \[^3^H\]-17\~-estradiol
(E~2~) is competing for binding to the ER with increasing concentrations
of either nonradiolabeled E~2~ (assay standard) or the coumarins. Each
datum represents the mean of at least 2 independent binding assays. The
competitor concentration at 50% reduction in \[^3^H\]-E~2~ binding
(IC~50~) is found at the intersection of the binding curves with the 50%
binding line ( ).

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 E~2~, 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 E~2~.

**FIGURE 6 **Competition curves for mycoestrogen binding to rat uterine
cytosolic estrogen receptor (ER). In this assay, \[^3^H\]-17\~-estra­diol
(E~2~) is competing for binding to the ER with increasing concentra­tions
of either nonradiolabeled E~2~ (assay standard) or the mycoestro­gens.
Each datum represents the mean of at least 2 independent binding assays.
The competitor concentration at 50% reduction in \[^3^H\]-E~2~ binding
(IC~50~) is found at the intersection of the binding curves with the 50%
binding line ( ).

**FIGURE 7 **Generalized molecular structures for the six categories of
chemicals examined. Ring labeling of carbon atoms allows identifi­cation
and location of substituent groups.

Chalcone, a nonhydroxylated chemical, exhibited weak binding to the ER,
i.e., 0.0015% that of E~2~. Monohydroxyla­tion of the chalcone structure
at the 4- or 4\~-carbons bound to ER with \~0.003% of the affinity of
E~2~, 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
\[^3^H\]-E~2~ 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 E~2~.

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 E~2~.
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 E~2~. Shifting these
hydroxyls to the \~ arrangement reduced ER binding to about 0.6% that of
E~2~ (\~-zearalanol) and 0.2% that of E~2~ (\~-zearalenol).

**DISCUSSION**

We used a competitive binding assay for the rat uterine ER, in which
receptor and ligand concentrations were optimized for chemical
throughput (26). Each assay included a complete competition curve for
E~2~ (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, E~2~. 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 E~2~ 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 ^IC^50^.^

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 \[^3^H\]-E~2~ bound at concentrations
above 85% of maximal com­petition. This biphasic curve suggests that very
high concen­trations of some competitors caused more \[^3^H\]-E~2~ to
bind to the ER. Borgna and Ladrech (34) showed that the dissociation
rate of the ER-\[^3^H\]-E~2~ complexes increases at high concentra­tions
of competitor. Although this would establish a different equilibrium for
\[^3^H\]-E~2~, this should result in a lowering of apparent
\[^3^H\]-E~2~ 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
\[^3^H\]-E~2~ 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
\[^3^H\]-E~2~ 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-\~ 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-\~.

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: E~2~, 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 E~2~, 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 estrogen­responsive reporter gene at a level
that is only 1.3% of that induced by E~2~.

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 ligand­specific 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 \[^3^H\]-E~2~ 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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