Inhibitory effects of Bifidobacterium-fermented soy milk on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinogenesis, with a partial contribution of its component isoflavones

Toshihisa Ohta 1 2 Seiichi Nakatsugi 2 Kouji Watanabe 2 Toshihiko Kawamori 2 Fumiyasu Ishikawa 1 Masami Morotomi 1 Shigeyuki Sugie 0 Toshiya Toda 3 Takashi Sugimura 2 Keiji Wakabayashi 2 0 Gifu University School of Medicine , 40 Tsukasa-machi, Gifu-shi, Gifu 500-8705 1 Yakult Central Institute for Microbiological Research , 1796 Yaho, Kunitachi-shi , Tokyo 186-8650 2 Cancer Prevention Division, National Cancer Center Research Institute , 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045 3 Fujicco Co. Ltd, 6-13-4 Minatojimanakamachi, Chuo-ku, Kobe, Hyogo, 650-8558, Japan 5To whom correspondence should be addressed Email: High consumption of soybean and soybean-related products is hypothesized to contribute to protection against breast cancer. Soybean is a rich source of genistein, a putative cancer chemopreventive agent. Fermented soy milk (FSM), which is made of soy milk fermented with the Bifidobacterium breve strain Yakult, contains larger amounts of the isoflavone aglycones genistein and daidzein than unfermented soy milk. In the present study, we examined the effects of FSM and its component isoflavone mixture (genistein:daidzein 4:1) on 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats. Starting at 7 weeks of age, female Sprague-Dawley rats were given PhIP at a dose of 85 mg/ kg body wt by intragastric administration four times a week for 2 weeks. They were fed control high fat basal diet or experimental high fat diet containing 10% FSM or 0.02 or 0.04% isoflavone mixture during and after carcinogen exposure. The incidences (percentage of rats with tumors) of mammary gland tumors were 71% in the control diet group, 51% in the FSM group and 68 and 61% in the groups treated with isoflavone mixture at 0.02 and 0.04%, respectively. Mammary tumor multiplicities (number of tumors per rat) were 1.2 0.2 for 10% FSM, 2.2 0.4 for 0.02% isoflavone mixture and 1.5 0.3 for 0.04% isoflavone mixture, being clearly smaller than the control diet value (2.6 0.5). Furthermore, feeding of FSM and the isoflavone mixture at both doses reduced the sizes of mammary tumors. Since the amounts of isoflavones in 10% FSM are approximately equivalent to those in the 0.02% isoflavone mixture, the chemopreventive activity of FSM could be partly attributable to the presence of isoflavones such as genistein and daidzein. - Breast cancer is one of the most common cancers in women. It accounts for almost 30% of all newly diagnosed malignant neoplasms in the USA (1). Several epidemiological studies have demonstrated that a high consumption of meat is associAbbreviations: FSM, fermented soy milk; HCAs, heterocyclic amines; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. ated with an increased risk of breast cancer (2,3). Moreover, the risk of breast cancer was found to be significantly elevated with an increased intake of well-done to very well-done meat (4). On the other hand, Gertig et al. reported that there was no association between meat intake or the cooking method of meat and the risk of breast cancer (5). Of the series of carcinogenic heterocyclic amines (HCAs) that have been identified in cooked meat, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), being the most abundant HCA in cooked foods (6), induces cancers in the mammary glands of female rats at a high incidence (79). Therefore, PhIP may play an important role in the development of breast cancer in humans. High consumption of soybean and soybean-related products has been suggested to contribute to a reduction in the risk of breast cancer in epidemiological studies (10). The average daily consumption of soybean and its products per person in Japan is much higher than the value for Americans (11,12). Many components, such as isoflavones, protease inhibitors, saponins and inositol hexaphosphate, have been investigated in the search for candidates responsible for the chemopreventive effects (13). Among these, isoflavones, particularly genistein, have been demonstrated to show several kinds of biological activity (14). Genistein inhibits protein tyrosine kinase and topoisomerase II activities (15,16). Genistein also has an inhibitory effect on angiogenesis (17), antioxidative potential (18) and phytoestrogenic activity (19). Among four isoflavones tested (genistein and daidzein and their -glucoside conjugates, genistin and daidzin), genistein was reported to most effectively inhibit the growth of LNCaP, a human prostate cancer cell line, while daidzein showed a weak inhibitory effect (20). The -glucoside conjugates genistin and daidzin exerted far less influence. Genistein can be absorbed in the upper small intestine (21), whereas the -glucoside conjugate genistin needs conversion to an aglycone through the action of a -glucosidase produced by intestinal bacteria before being absorbed. In general, the amounts of genistein and daidzein are much less than genistin and daidzin in soybean and its related products, such as soy milk and tofu (12). Based on biological activities of the isoflavone aglycones and glucoside conjugates and the conversion efficiency of glucoside conjugates to aglycones in vivo, soybean-related products containing higher amounts of aglycones than glucoside conjugates of isoflavones would be preferable for cancer prevention. Glycoside conjugates may be converted to aglycones by microbes during fermentation. Therefore, we produced fermented soy milk (FSM) with Bifidobacterium breve strain Yakult in an attempt to increase the amounts of isoflavone aglycones and also to improve the flavor of soy milk. Using the FSM thus obtained, inhibitory effects on PhIP-induced mammary tumor development in SpragueDawley female rats were examined. To determine the active principles in FSM, the effect of an isoflavone mixture of genistein and daidzein was also tested. Amount (g/kg diet) Materials and methods PhIP-HCl was obtained from the Nard Institute Ltd (Osaka, Japan). Flavone, used as an internal standard for the analysis of isoflavones, was obtained from Tokyo Kasei Co. (Tokyo, Japan). Genistein and daidzein, which were also applied as standard substances to estimate isoflavone contents, were purchased from Sigma Chemical Co. (St Louis, MO) and Seikagaku Corp. (Tokyo, Japan), respectively. Preparation of FSM and isoflavone mixture A seed culture of Bifidobacterium breve strain Yakult was added to soy milk (Shikokukakouki Co. Ltd, Tokushima, Japan) at 10 ml/l and fermentation was allowed to proceed statically at 37C for 18 h under anerobic conditions. The pH and viable cell counts of the FSM were 4.6 and 4.1 109 colony-forming units/ml, respectively. The FSM obtained was then lyophilized and levels of genistein, genistin, daidzein and daidzin in FSM were analyzed according to the method described by Kikuchi-Hayakawa et al. (22). These amounts were 1318 g for genistein, 26 g for genistin, 677 g for daidzein and 269 g for daidzin per gram lyophilized material. The crude prot (...truncated)