Isoflavone metabolites and their in vitro dual functions: They can act as an estrogenic agonist or antagonist depending on the estrogen concentration

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Abstract

The major soy isoflavones are daidzin and genistin, the glycoside conjugates of daidzein (DZ) and genistein (GTN). After ingestion, they are metabolized into diverse compounds in the gut. The marked inter-individual variation has been suggested in their metabolism. The clinical effects may be modulated by the metabolic ability to produce a more potent metabolite than the precursor. Our study was, therefore, designed to analyze and compare in vitro biologic activities of their metabolites: DZ, GTN, dihydrogenistein (DGTN), dihydrodaidzein (DDZ), tetrahydrodaidzein (TDZ), O-desmethylangolensin (ODMA), and equol (EQL). Furthermore, we investigated their modulatory effects in the presence of estrogen using several in vitro systems. The intermediate metabolites, such as DGTN, DDZ, and TDZ, bind much weakly to both ERs and induce less potently in transcriptional activity, gene expression, and mammary cell proliferation than their precursors. EQL has the strongest binding affinities and estrogenic activities especially for ERβ among the daidzin metabolites and shows the ability to suppress osteoclast formation at high doses. The test isoflavonoids act like estrogen antagonists with the premenopausal dose of E2 and thus inhibit estrogenic actions by E2, whereas they exert estrogen agonist activity with the lower dose of estrogen close to the serum levels of postmenopausal women. Our results suggest that phytoestrogens such as isoflavones may exert their effects as estrogen antagonists in a high estrogen environment, or they may act as estrogen agonists in a low estrogen environment.

Introduction

Phytoestrogens are a diverse group of plant-derived compounds that structurally and functionally mimic mammalian estrogens [1]. There are three major classes of phytoestrogens: the isoflavones, lignans, and coumestans. Of isoflavones, genistin and daidzin are the two most prevalent forms in soy foods. They are biologically inactive; once ingested, they are cleaved by glucosidases to “aglycones”, genistein (GTN) and daidzein (DZ) [2]. By intestinal microflora, GTN is further metabolized largely to non-estrogenic metabolites product, para-ethylphenol, while DZ is converted either to equol (EQL) or O-desmethylangolensin (ODMA) by two different metabolic pathways (Fig. 1) [3], [4], [5]. The new metabolites of DZ with additional hydroxyl groups have been recently identified but their levels were low in human urine [6], [7]. The metabolites undergo enterohepatic circulation after absorption. They can work in human body with the various estrogenic activities, often higher or lower than their precursors. There are some reports that show apparent inter-individual variation in the mode of metabolism with preference to EQL production in some individuals [5], [8], [9].

Phytoestrogens are known to have dual biologic functions; they can act estrogenically as estrogen agonists and antiestrogenically as antagonists [1]. The mechanism has not been completely clarified yet. A few in vivo data suggest that their biologic behavior may be modulated by the individual's amount of endogenous estrogens. Ingestion of a large amount of soy protein has been shown to suppress circulating female sex hormone levels and affect menstrual patterns in premenopausal women [10], [11]. Phytoestrogens, thus, may act primarily as an estrogen in a low-estrogen environment and as an estrogen competitor in a high estrogen environment.

Current evidence for the clinical usefulness of phytoestrogens is still controversial owing in part to their complex biological activity and the heterogeneity of the study populations, which results in individual metabolic variability. Well-characterized in vitro systems are needed to compare the diverse compounds including their metabolites for better understanding of their beneficial or adverse effects of an in vivo system on individual basis. We, therefore, analyzed and compared the in vitro biologic activities in a low or high estrogen environment. A few studies have been reported to evaluate estrogenic properties of isoflavones, which focused mostly on GTN or DZ [1], [12]. The biological characteristics of intermediate metabolites have not been fully assessed yet. Our study is first to determine the estrogen and antiestrogen activities of intermediate metabolites including tetrahydrodaidzein (TDZ), dihydrodaidzein (DDZ), and dihydrogenistein (DGTN).

Section snippets

Materials

17-β2-Estradiol (E2), 4-hydroxytamoxifen, hydroxyapatite (HAP), DZ (4′,7-dihydroisoflavone), and GTN (4′,5,7-trihydroxyisoflavone) were obtained from Sigma (St. Louis, Mo). Macrophage-cell stimulating factor (M-CSF) and soluble human receptor activator NFκ-B ligand (RANKL) were from Peprotech (Rocky Hill, NJ). Dual-Luciferase reporter assay system was purchased from Promega (Madison, WI). DGTN, DDZ, TDZ, EQL, and ODMA were synthesized and measured by liquid chromatography mass spectrometry

Results

Saturation ligand binding experiment with [3H]-E2 and linear transformation of saturation data revealed a single population of binding sites for E2 with Kd of 0.05 nM for the ERα and 0.08 nM for ERβ protein as shown in previous studies [16]. The binding affinities of the test compounds to the rhERα and β are shown in Table 1. All the compounds bound to both ER subunits. The relative binding affinity (RBA) of each compound was calculated as the ratio of concentrations of E2 and competitor required

Discussion

In our study, the estrogen agonist and antagonist properties of seven isoflavonoid derivatives were assessed in terms of their relative abilities to compete radiolabeled E2 for binding of ERα or β, to induce a transfected estrogen-responsive reporter gene and an estrogen target gene, to stimulate growth of the breast cancer cells, and to suppress osteoclast formation. The in vitro studies of phytoestrogens have focused especially on the biologic function especially of the two major soy

Acknowledgements

This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. (A060030). The authors appreciate Dr. Hyun-Ah Choi for proofreading the manuscript.

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