Evaluating estrogenic activity of isoflavones in miso using yeast two-hybrid method.

Abstract

Estrogenic activity in miso was evaluated without in vivo animal experimentation using in vitro method with yeast in a two-hybrid method because of its similarities with human cells. First, the recombinant yeast containing genes of human estrogen receptor (hER) was prepared for modeling human cells. Subsequently, standard solutions of 17β-estradiol and isoflavone (1.0 × 10-12 - 1.0 × 10-6 ) were assayed using the yeast. Their yeast produces β-glucosidase according to the concentrations of their solutions. Therefore, the estrogenic activity can be evaluated using recombinant yeast for the yeast two-hybrid method. Results show that 17β-estradiol has affinity to bind with Y187-αα. Genistein has affinity to bind with Y187-ββ. Daidzein, genistein, and glycitein in miso were 2.0-2.2 times the average concentrations of miso. Particularly, Mame miso had the highest concentration of isoflavones among all miso samples. Isoflavone in miso samples showed estrogenic activity against Y187-ββ. Mame miso had particularly high activity (1.97 U/OD660 1.0) against Y187-ββmodeling hERββ. Finally, the interaction of the human estrogen receptors was analyzed with 17β-estradiol and isoflavones using Y187 strains. Isoflavone inhibited the estrogenic activity of 17β-estradiol using Y187-αα.However, the estrogenic activity of 17β-estradiol against Y187-αβ and Y187-ββ, which model hER-αβandhER-ββ,was activated by isoflavone. Results showed genistein as the antagonist of estrogenic activity within 17β-estradiol against hERαα. However, it is an agonist of the activity within 17β-estradiol against hERαβ and hERββ. The yeast two-hybrid method has some potential for assessing the estrogenic activity of isoflavone in foods as a human model. PRACTICAL APPLICATION: Today, isoflavones in foods must be evaluated using in vivo methods such as animal experimentation because the estrogenic activities of isoflavones are agonist or antagonist with 17β-estradiol against estrogen receptors. Because animal experimentation is time-consuming and expensive, isoflavones in foods can be evaluated using yeast, a eukaryote that has similarity to human cells, while obviating in vivo methods. The yeast two-hybrid method is useful to assay the estrogenic activity of isoflavones in foods.