2007 Annual Report 1a.Objectives (from AD-416) 1. Induction, purification, and characterization of isoflavonoids in soybean seed, root, plant and cell culture incubated with A. sojae and A. sojae cell wall extracts. Induced isoflavonoids will be produced in soybean using several different biotic elicitors developed from A. sojae. Elicitor experiments will initially focus on soybean seeds, roots and cell cultures. Once an effective elicitor system is developed in the laboratory, elicitor experiments to induce isoflavonoids will be conducted on soybean seeds within the pod of a developing plant. 2. Determine hormonal and antioxidant activities of individual and combinations of induced isoflavonoids with in vitro bioassays. Determine hormonal and antioxidant activities of induced isoflavones purified under Objective 1 using in vitro bioassays. Three established assays for the determination of estrogenic and antiestrogenic activity will be utilized. Additionally, the antioxidant activities of induced isoflavones will be determined using three assays. 3. Determine hormonal and anticancer effects of individual and combinations of induced isoflavonoids with in vivo animal model systems. Determine estrogenic, antiestrogenic and anticancer activities of induced isoflavones (discovered in Objective. 1)in two in vivo mouse models. Induced isoflavones that display in vitro activity in Objective 2 will be lead candidates for in vivo testing. The glyceollins represent potential beneficial antiestrogens that need to be tested in animal models to confirm activity observed in vitro. Two different mouse model systems will be utilized to confirm estrogenic, antiestrogenic and anticancer activity (breast and ovarian cancer). 4. Develop methods for the organic syntheses of the glyceollins I, II and III. Also, precursors and synthetic analogues of the glyceollins will be produced and characterized for beneficial hormonal activities. Computer modeling methods will be developed to assist in the development of structure-activity relationships that point toward the most active forms of the molecule. Accomplishment of objectives 2-4 will require seeking out additional scientific cooperators with the particular expertise needed, either within ARS or in other organizations. 1b.Approach (from AD-416) Induced isoflavonoids will be produced in soybean using several different biotic elicitors developed from A. sojae. Elicitor experiments will initially focus on soybean seeds, roots and cell cultures. Once an effective elicitor system is developed in the laboratory, elicitor experiments to induce isoflavonoids will be conducted on soybean seeds within the pod of a developing plant. Isolated induced isoflavones will be tested for hormonal and antioxidant activities using several in vitro bioassays. Three established assays for the determination of estrogenic and antiestrogenic activity will be utilized. Additionally, the antioxidant activities of induced isoflavones will be determined using three assays. Induced isoflavones that display in vitro activity will be lead candidates for in vivo testing. The glyceollins represent potential beneficial antiestrogens that need to be tested in animal models to confirm activity observed in vitro. Two different mouse model systems will be utilized to confirm estrogenic, antiestrogenic and anticancer activity (breast and ovarian cancer). Also, organic syntheses methods for the glyceollins I, II and III will be developed. Precursors and synthetic analogues of the glyceollins will be produced and characterized for beneficial hormonal activities. Computer modeling methods will be developed to assist in the development of structure-activity relationships that point toward the most active forms of the molecule. 3.Progress Report This ARS research project is also the in-house project of the following cooperative agreements involved in like research: Tulane University/6435-42530-001-01G; Toledo University/6435-42530-001-02S; and Tulane University/6435-42530-001-03G. 4.Accomplishments Identification of the induced plant compounds glyceollins I, II, and III in soybean as therapeutic antiestrogens that inhibit the growth of estrogen-dependent cancers. -- Compared with the plant compounds genistein and daidzein, normally found in soy, purified glyceollins have displayed greater inhibition of estradiol effects on proliferation and estrogen receptor signaling in breast cancer cells. Glyceollins also are not strong plant estrogens which differs from the strong estrogens genistein and daidzein. These findings suggest that soy protein enriched in glyceollins may have distinct estrogen-modulating properties compared to standard soy protein. To test this idea, in the current study, a postmenopausal primate model was used to evaluate the short-term effects of glyceollin-enriched soy protein and standard soy protein isolate in combination with estrogen. Compared to baseline, estradiol treatment induced significantly greater breast proliferation in the control and soy protein group, but not in the glyceollin-enriched soy protein group. This change was accompanied by a reduction in estradiol-induced uterine area and markers of estrogen receptor alpha activity markers in the glyceollin-enriched soy protein group compared to the control group. These preliminary findings suggest that glyceollins may enhance the anti-estrogenic properties of standard soy protein isolate in reproductive tissues. This research is covered under Human Nutrition, National Program 107, Component 6: Health Promoting Properties of Plant and Animal Foods, Problem Statement 4.1.3: Develop more nutritious plant and animal products for human consumption. This past year, significant progress was made confirming the potential therapeutic value of the induced isoflavonoid glyceollins using an in-vivo (in laboratory test animals) model. -- We investigated the interactions between the induced soy phytoalexins glyceollins I, II, and III on the growth of estrogen-dependent MCF-7 breast cancer and BG-1 ovarian cancer cells implanted in mice with their ovaries removed. Treatment with glyceollin suppressed estrogen-stimulated tumor growth of breast cancer cells (-53.4%) and ovarian cancer cells (-73.1%) in mice with their ovaries removed. In contrast to the cancer agent tamoxifen, the glyceollins had no harmful effects on the uterine and partially blocked the effects of estrogen. These findings identify glyceollins as estrogen blocking agents that may be useful in the prevention or treatment of breast and ovarian carcinoma. This research is covered under Human Nutrition, National Program 107, Component 6: Health Promoting Properties of Plant and Animal Foods, Problem Statement 4.1.3: Develop more nutritious plant and animal products for human consumption. An HPLC method has been developed for isolating each individual glyceollin (I, II, and III). Small amounts (1-3 mg) of each glyceollin have been produced for in-vitro testing. Due to the difficulty in isolating large amounts of the glyceollins for experiments, a synthetic procedure was developed. The complete synthesis of glyceollins I and II has been completed. Small quantities (9 mg) of glyceollin I and (3 mg) of glyceollin II have been produced, and the process is being scaled up to produce large quantities necessary for animal studies. This research is covered under Human Nutrition, National Program 107, Component 6: Health Promoting Properties of Plant and Animal Foods, Problem Statement 4.1.3: Develop more nutritious plant and animal products for human consumption. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of invention disclosures submitted 1 Number of non-peer reviewed presentations and proceedings 4 Review Publications Wood, C.E., Clarkson, T.B., Appt, S.E., Franke, A.A., Boue, S.M., Burow, M.E., Mccoy, T., Cline, J.M. 2006. Effects of soybean glyceollins and estradiol in postmenopausal female monkeys. Nutrition and Cancer. 56(1):74-81. Salvo, V.A., Boue, S.M., Fonseca, J.P., Elliott, S., Corbitt, C., Collins-Burow, B.M., Curiel, T.J., Shih, B.Y., Carter-Wientjes, C.H., Wood, C.E., Erhardt, P.W., Beckman, B.S., Mclachlan, J.A., Cleveland, T.E., Burow, M.E. 2006. Antiestrogenic glyceollins suppress human breast and ovarian carcinoma tumorigenesis. Clinical Cancer Research. 12(23):7159-7164. Cole, R.B., Chou, C., Boue, S.M., Leblanc, B.W., Rodgers, A.H., Struck, R.F., Morgan, L.R. 2004. Gas chromatography-electron ionization mass spectrometry and liquid chromatography-electrospray tandem mass spectrometry for determination of impurities in the anti-cancer drug isophosphoramide mustard. International Journal of Mass Spectrometry. 231:147-155.