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United States Department of Agriculture

Agricultural Research Service

2006 Annual Report

1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Hormonal changes in post-menopausal women can increase the risk of developing breast cancer, bone loss, and reduced cardiovascular health. Populations consuming a diet high in soybean phytoestrogens (that mimic human estrogen) have lower incidences of several diseases, including breast and prostate cancer. Recently, research has shown that isoflavones, a group of phytoestrogens found in soybean derived foods, are compounds that reduce the risk of certain potential health problems when consumed in the diet. Identifying the phytoestrogenic activity of soybean isoflavones would benefit the nutritional health of the population in general, particularly women. Techniques are being determined by the Agricultural Research Service (ARS) scientists at the Southern Regional Research Center (SRRC) to manipulate phytoestrogen levels in soybean seed and soy-based products to maximize health benefits by their consumption. Several biotic (living) and abiotic (non-living) elicitors have been evaluated for potential use in effectively inducing beneficial isoflavones (plant compounds). Collaborating scientists from Tulane University have performed a series of in vitro (laboratory) bioassays using various phytoestrogen compounds extracted from elicitor-treated soybean seeds with higher levels of certain phytoestrogens. Based upon preliminary results, these inducible phytochemicals demonstrated the potential to act as protective agents in vivo (in laboratory test animals) against hormone-dependent cancers, indicating their possible use as anti-cancer “nutraceuticals” (medicines from plants) in humans once threshold levels are determined. The probability that an American woman will develop breast cancer is currently estimated to be one in nine. Evidence suggests that natural steroidal estrogens (xenoestrogens), or compounds that mimic the biological activities of natural steroidal estrogens, are encountered in industrial environments where incidence of breast cancer has been on the rise. The occurrence of breast cancer is lower, by comparison, in Asian women who consume much greater quantities of legume products, including soybeans that contain isoflavonoids. The research project relates to National Program 107, Human Nutrition. Research to manipulate and produce optimal levels of beneficial “nutraceutical” compounds (such as phytoestrogens) in soybean in the field and in resultant soy-based products will:.
1)add value to soy-based products, thus benefiting the agricultural economy in the U.S. and.
2)increase the health of females consuming soy-based products, thus helping to decrease health problems and costs due to pre- and post-menopausal hormone changes.

2.List by year the currently approved milestones (indicators of research progress)
FY 2006: Milestone 1: Complete preparation of a cell wall extract elicitor from A. sojae.

Milestone 2: In vitro testing of glyceollin mixture (I, II and III).

Milestone 3: In vitro testing of induced soybean extracts.

Milestone 4: Complete synthesis of glyceollin I.

Milestone 5: Complete assembly of computational models for ER alpha and ER beta receptors.

FY 2007: Milestone 6: Complete production of induced isoflavones using cut-cotyledon assay and soybean root assay.

Milestone 7: Complete identification and quantitation of isoflavones in soybean tissues and cell cultures using HPLC; Isolation of glyceollin mixture.

Milestone 8: In vitro testing of glyceollin mixture (I, II and III).

Milestone 9: In vitro testing of individual glyceollins I, II and III.

Milestone 10: In vivo testing of a mixture of glyceollins subcutaneously.

Milestone 11: Complete synthesis of glyceollins II and III.

FY 2008: Milestone 12: Complete isolation of glyceollins I, II and III.

Milestone 13: Complete production of induced isoflavones using cell cultures.

Milestone 14: In vivo testing of a mixture of glyceollins orally.

FY 2009: Milestone 15: Complete isolation of novel induced isoflavones.

Milestone 16: In vivo testing of glyceollin I, II and III.

Milestone 17: Utilize models to assist in understanding biological activity and to optimize design of glyceollin analogs.

FY 2010: (This project is scheduled to end in FY 2009, and will be extended 1 year into FY 2010.) Milestone 18: In vitro testing of glyceollin analogs/precursors.

Milestone 19: In vitro testing of glyceollin precursors and analogs.

Milestone 20: In vivo testing of glyceollin precursors and analogs.

Milestone 21: In vivo testing of glyceollins I, II, III and glyceollin precursors/analogs.

Milestone 22: Produce multi-gram quantities of the most promising glyceollin and/or analogs/precursors.

4a.List the single most significant research accomplishment during FY 2006.
Therapeutic Effects of the Glyceollins: This past year, significant progress was made confirming the potential therapeutic value of the induced isoflavonoid glyceollins using in vivo models. Compared with the isoflavones 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 have enhanced antagonism toward estrogen receptor alpha relative to beta and lack any of the estrogen agonist activity of genistein and daidzein seen in low estrogen conditions. 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. Project scientists also tested the hypothesis that specific glyceollins, isolated from elicited soy, would display anti-estrogenic activity, suppressing the proliferation of estrogen receptor-positive estrogen dependent breast cancer cells and inhibiting estrogen receptor-dependent gene expression. To this end, project scientists have established the ability of glyceollins to significantly suppress tumor growth of MCF-7 cells in female nude mice. Additionally, using MCF-7 breast cancer cells as a model of anti-estrogen resistant breast carcinoma, project scientists demonstrated the ability of glyceollins to antagonize cancer cell mediated colony formation and tumor growth as compared to the anti-cancer agent tamoxifen. Taken together, the results established the in vivo suppression of estrogen-dependent and independent tumor growth by glyceollins (I-III), while also providing critical information in a number or areas directly applicable to the understanding and treatment of breast carcinoma. (1) Glyceollins may represent the parent structure for a new class of anti-estrogen that would fill a critical need in the therapy of breast carcinoma. (2) The glyceollins may represent important components of a soy or enriched soy-based diet in terms of chemoprevention. (3) The glyceollins may represent compounds useful in the treatment of tamoxifen-resistant breast cancer or other hormone dependent cancers (endometrial, prostate, ovarian).

IMPACT: This research could lead to a better understanding of the effects of induced phytoestrogens on human health and may lead to alternative or value-added uses of induced phytoestrogens, including the therapeutic use of the glyceollins as anti-cancer agents.

4b.List other significant research accomplishment(s), if any.
Induced Isoflavones From Soybean Cells: A soybean suspension cell culture was developed and utilized for the characterization of elicitors. The ability of several elicitors at inducing phytoalexins in soybean liquid cell culture was evaluated. Of particular interest was the ability of each elicitor to induce the isoflavone phytoalexin glyceollin, which has recently been shown to be anti-estrogenic in several in vitro assays. Soybean cells from both control and elicitor-exposed cultures were analyzed using High Performance Liquid Chromatography (HPLC)-Mass Spectrometry (MS). Several constitutive isoflavones have been identified and preliminary data indicate novel phytoalexins related to the glyceollins are formed. Results have shown that a cell wall extract derived from Aspergillus flavus and Aspergillus sojae produced higher levels of the glyceollins.

IMPACT: This research will lead to a better method for producing novel isoflavones in soybean. These isoflavones could benefit human health and may lead to alternative or value-added uses of induced phytoestrogens, including the therapeutic use of the glyceollins as anti-cancer agents.

Isolation of Glyceollins I, II and III: An HPLC method has also 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.

IMPACT: This research will lead to complete characterization of the glyceollins, and a better understanding of the effects of induced phytoestrogens on human health, and may lead to alternative or value-added uses of induced phytoestrogens, including the therapeutic use of the glyceollins as anti-cancer agents.

Synthesis of Glyceollin I: A synthetic route for the production of glyceollin I has been developed. Small quantities (1-2 mg) of glyceollin I have been produced, and the process is being scaled up to produce large quantities necessary for animal studies.

IMPACT: This research could benefit farmers and researchers developing methods to eliminate aflatoxin from oilseed crops.

4c.List significant activities that support special target populations.

4d.Progress report.

5.Describe the major accomplishments to date and their predicted or actual impact.
This project is a continuation of ARS project 6435-4200-017-00D entitled “Identify Mechanisms of Isoflavonoid Induction in Legumes and Their Phytoestrogenic Effects,” and is the ARS parent project of the following cooperative agreements involved in like research: McLachlan/Tulane/6435-42530-001-01G and Erhardt/University of Toledo/6435-42530-001-02S. In addition to the accomplishment described under Question 4, this project has also accomplished the following: 1. Nearly 100 flavonoid compounds were supplied to Tulane University where they were tested in animal system bioassays for phytoestrogenic (estrogenic or anti-estrogenic) activities. 2. Several of the compounds showed estrogenic activity in an easily measured, laboratory “activity reporter” type assay (which involves use of a cloned estrogen receptor gene-reporter bioassay in yeast), and the breast cancer cell (MCF-7 class) proliferation bioassay. 3. Recent results indicated that glyceollins and other phytoalexins (plant compounds induced in response to elicitors) in the isoflavonoid chemical class from soybean have anti-estrogenic activity. The implication is that exposure to pathogen attack or other plant stresses may influence potential phytoestrogenic compounds in soybean in the field. 4. These findings have led to the isolation of other induced plant compounds from different legumes for phytoestrogenic analyses, including phaseollin (plant compound) from green beans and pisatin (plant compound) from snow peas. 5. Critical chemical side chains of these molecules were identified that are required for estrogenic activity, and preliminary results indicate several of these functional side chains are necessary for anti-estrogenic activity. The research showed that through use of naturally occurring phytoestrogens comprising a diversity of chemical structures, important data on structure-activity relationships can be obtained. 6. ARS scientists have identified several fungal inducers (elicitors), including A. sojae, which induce phytoestrogen levels in soybean cotyledons (seeds) several fold over non-induced cotyledons. Several abiotic (non-living) elicitors have been utilized for the induction of isoflavonoid phytoalexins; however, research has focused on non-toxic microbial based elicitor systems. These experiments demonstrate precedence for our goals to: a) manipulate phytoestrogen levels in soybean and soybean products for maximal health benefits and b) hyperinduce phytoestrogen biosynthetic pathways to identify temporary intermediates and new trace compounds which may also have phytoestrogenic activities.

IMPACT: The impact of this research is that the identification of beneficial plant compounds in the isoflavone, isoflavonoid and flavonoid class of chemicals, both those that are induced by certain treatments (e.g. with living microbes or elicitor chemicals derived from microbes) and/or those that are constitutive (already present) in plants, could lead to benefits in human health. Technology to increase beneficial isoflavonoids in soybean (other legumes) could be implemented through genetic manipulation of the plant in the long run, or through the use of elicitor compounds derived from microbes that could be sprayed on plants or seeds to increase beneficial phytoestrogens. Other potential benefits of the research to identify and increase production of isoflavonoids are explained in Questions 1 and 2, above.

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
The ability to elicit isoflavonoids in soybean and other legumes is a well established technique that was first examined as a defensive mechanism of plants. The potential of these induced isoflavonoids as therapeutic compounds has not been previously investigated. One patent application on the use of the glyceollins as therapeutic agents is currently pending. This science is being made available to other scientists in both industry (Solae Corporation) and academia (Tulane University, Xavier University, University of Toledo and Wake Forest University). Certain technological obstacles have to be overcome before a viable food product is produced. Potential Constraints:.
1)the degree of industry willingness to adopt new production/processing methods to enhance isoflavonoid content of existing products;.
2)availability of germplasm adapted for the region if induction methods are found to be optimal only through using specific, perhaps exotic soybean varieties or types;.
3)public willingness to accept/consume products that may have been enhanced in isoflavonoid content using application of microbes or derivative elicitor chemicals from the microbes; and.
4)increasing isoflavonoid compounds above optimal levels in seed may be problematic, since some evidence suggests that these compounds may be responsible for disagreeable flavor qualities in many soybean products.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).

Last Modified: 10/21/2014
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