Comparative study of estrogenic activities of phytoestrogens using OECD in vitro and in vivo testing methods

With growing scientific interest in phytoestrogens, a number of studies have investigated the estrogenic potential of phytoestrogens in a wide variety of assay systems. However, evaluations of individual phytoestrogens with different assay systems make it difficult for predicting their relative estrogenic potency. The objective of this study was to compare estrogenic properties of fifteen known phytoestrogens using an estrogen receptor-α (ER-α) dimerization assay and Organization for Economic Cooperation and Development (OECD) standardized methods including in vitro estrogen receptor (ER) transactivation assay using VM7Luc4E2 cells and in vivo uterotrophic assay using an immature rat model. Human ER-α dimerization assay showed positive responses of eight test compounds and negative responses of seven compounds. These results were consistently found in luciferase reporter assay results for evaluating ER transactivation ability. Seven test compounds exhibiting relatively higher in vitro estrogenic activities were subjected to uterotrophic bioassays. Significant increases in uterine weights were only found after treatments with biochanin A, 8-prenylnaringenin, and coumestrol. Importantly, their uterotrophic effects were lost when animals were co-treated with antagonist of ER, indicating their ER-dependent effects in the uterus. In addition, analysis of estrogen responsive genes revealed that these phytoestrogens regulated uterine gene expressions differently compared to estrogens. Test methods used in this study provided a high consistency between in vitro and in vivo results. Thus, they could be used as effective screening tools for phytoestrogens, particularly focusing on their interactions with ER-α.