Analytical, Nutritional and Clinical Methods A two-step screening method, using estrogen receptor-mediated transactivation, to measure estrogenicity in edible plants Yukitomo Arao c, * , Namiko Kanamori a , Eri Kikkawa b , Hiroko Otsuka a , Yasushi Arimoto b , Kazuhiro Ikeda a , Takahiro Inakuma b , Fujio Kayama a,c,d, * a Department of Environmental Medicine, Center for Community Medicine, Jichi Medical University, Shimotsuke-shi, Tochigi 329-0498, Japan b Research Institute, Kagome Co., Ltd., Nasu-gun, Tochigi 329-2792, Japan c Laboratory of Reproduction and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, United States d CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan Received 10 May 2004; received in revised form 10 January 2007; accepted 10 January 2007 Abstract Estrogenic activity in 88 edible plants was screened using a human ovarian carcinoma cell line stably transformed with estrogen- responsive elements (ERE) fused to a luciferase (luc) reporter gene (BG1Luc4E(2)). We found 18 plants (ashitaba, avocados, chinese mustard, chinese chive (yellow), chrysanthemum, dokudami, shantung greens, green soybeans, soybean seeds, soybean sprouts, hop, jap- anese pepper, kidney beans, kuromame, perilla, peas (immature), plantain, and pomegranate juice) expressing estrogenic activity in BG1Luc4E(2) cells. To confirm that the phytoestrogenic activity occurred via estrogen receptors (ER), the reporter vector (ERE-tk- luc) and an expression vector, containing either ERa or ERb, were used to transiently transfect 293T cells. Extracts from avocados, plan- tain and dokudami did not activate ERa- and ERb-mediated transcription. In conclusion, we report a simple and quick screening method for phytoestrogenic activity in plant extracts using BG1Luc4E(2) cells and confirmation of the results by ERa- or ERb-trans- fected 293T cells. This two-step screening method has a practical application in screening estrogenic substances in edible plants. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Phytoestrogen; Estrogen receptor; Transcription; Screening 1. Introduction In the recent literature, there have been many reports on plants containing a substance called phytoestrogen that exerts estrogen-like activity in animals (Safe, Connor, Ramamoorthy, Gaido, & Maness, 1997). Phytoestrogens are believed to induce developmental changes in endocrine, nerve and immune systems in animals (Jefferson & New- bold, 2000). Epidemiological and experimental studies sug- gest that phytoestrogens, not only possess estrogenic activities, such as uterine growth and inhibition of bone loss (Ishimi et al., 2000), but also anti-estrogenic activities, such as growth inhibition of mammary carcinoma cells (Fioravanti et al., 1998; Miodini, Fioravanti, Fronzo, & Cappelletti, 1999). There is limited information on which foods contain estrogenic or anti-estrogenic activity so it is important to screen for phytoestrogenic activity in edible plants in order to evaluate beneficial or adverse human health effects. 0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2007.01.076 * Corresponding authors. Address: Laboratory of Reproduction and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, United States. Tel.: +1 919 541 7932; fax: +1 919 572 9825. E-mail addresses: araoy@niehs.nih.gov (Y. Arao), kayamaf@jichi. ac.jp (F. Kayama). www.elsevier.com/locate/foodchem Food Chemistry 104 (2007) 1288–1294 Food Chemistry