The human estrogen receptor (hER) exists as two subtypes, hER a a and hER b b, that differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activities of soy isoflavones after digestion with enteric bacteria in competition binding assays with hER a a or hER b b protein, and in a gene expression assay using a yeast system. The estrogenic activities of these isoflavones were also investigated by the growth of MCF-7 breast cancer cells.Isoflavone glycoside binds weakly to both receptors and estrogen receptor-dependent transcriptional expression is poor. The aglycones bind more strongly to hER b b than to hER a a. The binding affinities of genistein, dihydrogenistein and equol are comparable to the binding affinity of 17 b b-estradiol. Equol induces transcription most strongly with hER a a and hER b b. The concentration required for maximal gene expression is much higher than expected from the binding affinities of the compounds, and the maximal activity induced by these compounds is about half the activity of 17 b b-estradiol. Although genistin binds more weakly to the receptors and induces transcription less than does genistein, it stimulates the growth of MCF-7 cells more strongly than does genistein.

The intake of weakly estrogenic isoflavonoids (phytoestrogens) is high in countries with a low incidence of estrogenrelated cancers, such as breast and prostate cancers. 1) Two ERs (a and b ) have been identified to date and the physiological responses of estrogens are known to be mediated within specific tissues by at least these two receptors.2,3) The ERs are a 3A member of the nuclear hormone receptor family and act as a ligand-activated nuclear transcription factor. 4)In a previous paper, we reported the results of systematic examination of the estrogenic activities of soy isoflavones (e.g. daidzin, genistin and glycitin) and their metabolites (e.g. daidzein, genistein, glycitein, equol, dihydrogenistein and dihydroglycitein) by enteric bacteria, 5) and the activities of several other isoflavone derivatives isolated from Pueralia lobata and P. thomsonii. 6)Usually, when soy foods are consumed, the soy isoflavones are metabolized into their aglycones and the related compounds by enteric bacteria. We have proved in in vitro experiments that the soy isoflavone glycosides are less estrogenic than the metabolites, i.e., they are activated by enteric bacteria. The metabolites produced by enteric bacteria are absorbed through the intestinal membranes and transported to the liver where they undergo re-metabolization by hepatic enzymes. To evaluate the bioactive compounds in soy products, investigation of the compounds which are actually absorbed within the body is necessary. 7,8) Herein, we describe the results of an investigation on the estrogenic activities of soy isoflavone metabolites isolated from human urine samples. Their effects on the estrogen dependent growth of MCF-7 cells, 9,10) on human ERs (hERs a and b )-dependent b-galactosidase induction, and their binding behavior to hERs were studied. We also examined the estrogenic activities of some synthetic isoflavone sulfates for the purpose of comparison. [2,4,6, H(N)]-17b-Estradiol (72 Ci/mmol) was purchased from Dai-Ichi Pure Chemicals Co. Ltd. RPMI 1640 medium (with or without phenol red), fetal bovine serum (FBS), trypsin/EDTA, Dulbecco's phosphate-bufferd saline (PBS), and kanamycin sulfate were purchased from Lifetech (Rockville, MD, U.S.A.). MATERIALS AND METHODS ChemicalsIsoflavone Metabolites Isoflavone metabolites examined in this work are shown in Fig. 1. M-1-9 were isolated from human urine samples collected for 12 h after feeding soy bean curds.11) Dihydrodaidzein and O-desmethylangolensin (O-DMA) were synthesized following the method described by Wahara et al.12) Genistein 4Ј-O-sulfate (S-1), 4Ј,7-di-Osulfate (S-2), daidzein 4Ј-O-sulfate (S-3), and 4Ј,7-di-O-sulfate (S-4) were synthesized following the method described by Peterson et al. 13)Cells MCF-7 cells were supplied by the Cell Resource Center for the Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University.Growth of MCF-7 Cells MCF-7 cells were cultured in phenol red-free RPMI 1640 medium supplemented with 10% (v/v) FBS and kanamycin sulfate (100 mg/ml) in 25 cm...

Estrogens play important hormonal roles in all vertebrates. Animal estrogens are exclusively steroidal compounds, and the principal physiological estrogen in most species is 17b-estradiol. Many plants produce isoflavones that possess estrogenic activity in animals and are, thus, called phytoestrogens. Two estrogen receptors (ERs) have been identified to date 1,2) and the physiological responses to estrogen are known to be mediated within specific tissues by at least these two receptors. The ERs are a 3A member of the nuclear hormone receptor family and act as ligand-activated nuclear transcription factors. 3)Among the foods consumed by humans, soybeans contain the highest concentration of isoflavones. We have examined the estrogenic activity of these soy isoflavones (e.g., daidzin, genistin and glycitin) and their metabolites (e.g., daidzein, genistein, glycitein, equol, dihydrogenistein and dihydroglycitein) by enteric bacteria in the previous paper. 4) In this paper, we examined the estrogenic activities of several other isoflavone derivatives by (A) binding to human estrogen receptor (hER) a and b, and (B) effect on estrogen receptordependent transcriptional expression. 4) MATERIALS AND METHODS

We synthesized fifteen oligopeptides consisting of Asp or Glu conjugated with a fluorescent probe, 9- fluorenylmethylchloroformate (Fmoc). In the in vitro binding assay to putative hydroxyapatite (HA), the affinities of these conjugates depended only on the number of amino acid residues, not on their optical characters (L or D) or their species (Asp or Glu). In an in vivo experiment involving a single i.v. injection of Fmoc-D-Asp oligopeptides into mice, peptides consisting of over six Asp residues were selectively distributed to the bone. Then, we synthesized estradiol-17 beta-succinate-(L-Asp)6 [E2-(L-Asp)6] and studied its pharmacokinetic characteristics and its antiosteoporotic effects on ovariectomized (OVX) mice. Although the distribution volume of E2-(L-Asp)6 was significantly smaller than that of E2, E2-(L-Asp)6 was selectively distributed in the bone after i.v. injection and gradually decreased during 7 days. E2-(L-Asp)6 effectively prevented OVX-induced bone loss, without altering the uterine weight, in the dosage range of 0.11 to 1.1 mumol/kg once a week, while E2 increased both the bone mineral density and uterine weight at 0.37 mumol/kg every third day. The results suggest that acidic oligopeptide may be useful for drug delivery to bone and E2-(L-Asp)6 is a good candidate as an anti-osteoporosis drug without the adverse side effects of E2.

Benzo[a]pyrene (BaP), a major environmental pollutant, is metabolized in vivo and produces many hydroxy derivatives. The estrogenic/antiestrogenic activities of twelve monohydroxy derivatives of BaP (1-through 12-OH species) were investigated using competition binding to human estrogen receptor (hER)α and hERβ, and the gene expression assay of the yeast two-hybrid system. BaP and 5-OH BaP did not bind to either hER. The other monohydroxy derivatives bound to both hERs. These compounds bound more strongly to hERβ than to hERα. Using the yeast two-hybrid assay system, 1-, 2-, 3-, and 9-OH BaP induced β-galactosidase with hERβ but not with hERα. This suggested that these compounds were estrogenic. In the presence of 10 -9 M 17β-estradiol, 8-OH BaP inhibited the induction of β-galactosidase. Because 8-OH BaP did not affect cell growth, it appeared to be an antiestrogen. The present study shows that most of the monohydroxy derivatives of BaP bind to estrogen receptors (ERs), and several of them have estrogenic or antiestrogenic activity.