Duration: 10/01/2002 to 09/30/2007

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Overview. This application represents a renewal of a regional project that has been in existence since 1971. The project focused initially on the identification of food-borne natural toxicants of specific interest to the Western region. W-122 now targets food safety and health issues that are important to all Americans. Research under W-122 continues to address foodborne toxicants, both natural and induced, but has evolved to include bioactive dietary constituents including anti-carcinogens, anti-toxicants, phytoestrogens, immune modulators and antimicrobials. A unique and critical feature of W-122 is its focus on understanding the molecular basis of how bioactive dietary chemicals impact human health that is undergirded by a firm grounding in state-of-the art analytical chemistry. These efforts, which involve cell culture, animal models and human epidemiological studies, cover a spectrum of cutting edge scientific approaches that include molecular signaling, DNA damage, genomics, tumor formation, and intermediate biomarkers of cancer. The overall objectives of this renewal application continue within W-122's established theme, however, specific experimental questions and methodologies have evolved and experiment station participants have changed or been expanded. W-122 has been highly successful in meeting its objectives, as measured by its numerous collaborative efforts, and it is anticipated that the renewal will be equally successful.

Issues related to food safety and to diet and health impact many stakeholders. The food supply in the U.S. is regarded as among the world's safest, having high nutritional quality and extremely low carryover of agricultural chemicals. Advances in food processing help to provide a high measure of safety against many potential toxic and microbiological hazards. Nevertheless, through their interactions with the public and agrifood industry, W-122 members have determined that an issue of paramount concern to stakeholders is the relationship between dietary chemicals and human health. Stakeholders include consumers, agricultural producers, food processors, health professionals, and policy makers charged with maintaining a safe and nutritious food supply.

One aspect of these stakeholders' concern is the potential of bioactive dietary chemicals to cause disease. For example, certain fungal toxins can mediate cancer in a genotoxic fashion by direct DNA damage or in an epigenetic fashion by tumor promotion. Indeed, excluding smoking, diet-related factors would account for over 50% of all remaining cancer deaths in this country, or 150,000 food-related cancer deaths per year in the U.S. alone (National Research Council, 1996). Thus, the relationship between specific dietary components and cancer clearly needs to be better understood. In addition to cancer, it is well-known that dietary chemical toxicants can cause metabolic dysregulation, gastroenteritis, neurotoxicity, immunotoxicity, and reproductive toxicity in animals which are potentially of immense human concern.


Conversely, a second aspect of these stakeholders' concern is growing evidence that many dietary bioactive dietary chemicals can improve human health. For example, epidemiologic studies indicate that regular consumption of several food commodities, especially fruits and green and yellow vegetables rich in anticarcinogenic factors, correlate negatively with several human cancers. Thus, a more realistic view of the diet-cancer relationship is that variation in cancer rates reflects the presence or absence of natural chemoprotective factors in an individual's daily diet as well as the level of food-borne carcinogens and tumor promoters. As a specific example, chlorophyll and its derivatives can function as anticarcinogens and block the action of genotoxic chemicals such as aflatoxin. Bioactive dietary chemicals can affect other chronic diseases. Omega-3 fatty acids which are produced by plants and can be concentrated in certain fish species are well-known to promote cardiac health. Furthermore, many herbal supplements contain chemicals which have anti-inflammatory, immune stimulatory and neuroendocrine properties.

A third aspect of the stakeholders' concern is the often baffling complexity associated with separating the positive and negative attributes of a dietary chemical. Notably, dosage and timing of exposure can determine whether these bioactive compounds are beneficial or detrimental. Additionally, certain populations may be more sensitive regarding undesirable effects. For example, the dietary estrogens may produce beneficial effects relative to cardiovascular disease but undesirable effects in postmenopausal women with estrogen-responsive breast cancer. Similar paradoxical effects can be observed with many biologically active dietary components.

Importance of Studying Dietary Bioactive Chemicals. Bioactive dietary chemicals are extraordinarily diverse with respect to chemical structures and biological activities. There are numerous kinds of dietary toxicants and the mycotoxins, structurally unrelated metabolites of molds, illustrate this diversity. Aflatoxin is a potent carcinogen often found in U.S. foods such as corn, peanuts and milk. Recent studies indicate that fumonisin contamination of the U.S. corn supply may represent an even greater hazard due to its carcinogenic and teratogenic properties. Wide-spread Fusarium mold contamination of wheat and barley and resultant vomitoxin contamination in 1996 and 2000 prompted food/cereal companies and the brewing industry to stop purchasing these commodities from the Midwestern U.S. Mycotoxins are not only carcinogens, but can suppress immune responses, rendering the consumer more susceptible to adventitious infections. The presence of mycotoxins in agricultural crops is further complicated by their transfer from feed to animal tissue which further dispenses them in the food supply. Going beyond the mycotoxin example, numerous other toxic natural metabolites and anthropogenic exist in our food.

Brassica indoles and chlorophyl are examples of beneficial dietary chemicals. These compounds can act as cancer preventative agents. Indoles are strong inhibitors of chemical induced mammary tumors and spontaneous uterine tumors in rodents. Chlorophyl and it derivatives have the potential to reduce cancer risk by sequestering active carcinogens in the gastrointestinal tract which reduced carcinogen exposure. Risk of liver, colon, stomach, and lung cancer which include the leading causes of cancer death in the US and the world, may easily be cut in half by appropriate daily intake of simple, safe, inexpensive chlorophyll derivatives. Whether the native chlorophylls in green vegetables may have similarly protective benefits is not known, and we intend to pursue this. Failure to carry out this work would represent a lost opportunity to have a major, world-wide influence in reducing cancer risks.

Other examples of beneficial dietary chemicals are the omega-3 fatty acids, certain herbal supplements and prebiotics which may affect the immune system and be particularly important in the prevention and treatment of autoimmune and inflammatory diseases. Examples of target diseases include rheumatoid arthritis multiple sclerosis, immune-mediated or type 1 diabetes mellitus, inflammatory bowel diseases, systemic lupus erythematosus, psoriasis, scleroderma, and autoimmune thyroid diseases. These diseases can afflict millions of Americans. Typically autoimmune diseases strike women more often than men and notably, they impact women of working age and during their childbearing years. Certain autoimmune diseases occur more frequently in certain minority populations. Lupus is more common in African-American and Hispanic women than in Caucasian women of European ancestry. Rheumatoid arthritis and scleroderma impact a higher percentage of residents in some Native American communities than in the general U.S. population. Clearly, failure to exploit the potential for bioactive dietary chemicals to ameliorate such diseases would have far-reaching social, economic, and health impact.


In some cases, bioactive dietary chemicals have paradoxical effects. The soy phytoestrogen, genistein, has been shown to reduce the number of DMBA-induced mammary tumors when administered early (prior to puberty). The likely mechanism responsible was an estrogenic effect of genistein which caused an enhancement of mammary gland differentiation and a reduction in mammary cell proliferation which ultimately reduced the amount of DNA damage from the DMBA. This presents a paradox because an estrogenic effect is associated with enhancement of estrogen-dependent breast cancer growth. The dietary estrogen, genistein, which has been shown to enhance growth of estrogen-responsive breast tumors is being investigated by W-122 researchers.

Of particular relevance to this project, there is a burgeoning herbal product and food supplement industry in the U.S., recently estimated at $20 billion-a-year (2002). One reflection of public interest in this industry was passage of the Dietary Supplement Health and Education Act by the U.S. Congress in October 1994. With proper control and monitoring, identification and verification of effective herbal products could reduce medical costs and possibly provide new agricultural commodity opportunities. On the other hand, lack of quality control and a blurring of boundaries among functional foods, food supplements (vitamins, antioxidants, etc.) and medicinal herbs has sometimes allowed toxic substances to enter the human food chain. In addition to individual toxicity problems, these occurrences may reflect negatively on truly useful products. Additionally, such agents have the potential to interfere with successful therapies or medications. For example the dietary estrogen, genistein can negate the inhibitory effect of tamoxifen on estrogen-stimulated breast cancer growth. Other examples include the ability of Saint John's wort to act in an additive manner with anti-depressant drugs.

Dietary bioactive chemicals are defined in this proposal as naturally occurring substances in plants or other food products that exert beneficial or undesirable effects when they are consumed. The focus of W-122 is on endogenous plant or animal metabolites, compounds derived from the growth of fungi and algae on foodstuffs, and substances created (induced) during cooking or other processing of the food. An additional category of induced bioactive components are substances that may appear inadvertently in genetically manipulated plant materials which result from efforts to alter plant quality. This proposal will continue to focus attention on the role of food-related anticarcinogens as chemopreventive agents for reducing human cancer, and also on natural beneficial compounds which improve health and reduce chronic disease. This proposal also seeks to identify, understand, and eliminate, in so far as practicable, the specific toxicants in the food supply that contribute to health deficits.

Technical Feasibility of Studying Bioactive Dietary Chemicals. The work is viable because of major recent technical and conceptual advances in the fields of molecular and cell biology related to cancer and other chronic diseases. New knowledge of the regulatory pathways for cell cycle, cell differentiation and programmed cell death and their relationship to disease now allows the identification and exploration of cellular control mechanisms. W-122 members have a long track record of funding and publications dealing with mechanisms and hazards of dietary toxicants as well as exploitation of beneficial dietary substances. Analytical methods are well established to do the proposed work and specific advances and contributions have already been made relative to novel bioactive chemical isolation, identification, and bioassay. W-122 scientists have extensive experience in model systems (cell culture, transgenic and knockout mice, rats poultry, rainbow trout, human subjects) with which to pursue this work, and have published all the data necessary for proof-of-concept work that demonstrates the benefits of chlorophylls, indoles, tea anti-oxidants, and omega-3 fatty acids as well as the efficacy of various antimicrobials.


Advantages of Multi-state Study of Bioactive Dietary Chemicals. Defining the role of bioactive dietary chemicals in cancer and chronic disease is an exceedingly complex undertaking. The problems to be addressed and benefits to be achieved are region- or nation-wide and not confined to a single state. Thus, no single research group or experiment station has all the expertise and facilities to fully address the issues involved for even one protective or risk factor. The proposed work requires collaboration from those with diverse academic backgrounds (toxicology, molecular biology, nutrition, risk assessment, analytical chemistry) as well as geographic diversity due to the wide range of food crops that may be involved. W-122 members have an established history of collaboration on bioactive dietary chemical research, and the collective expertise and facilities to continue to make great progress. Since several of the issues are being addressed in different ways among researchers in several states, information exchange and collaborations will both enhance reaching the goals and limit duplication of effort among members' respective units. Their positions as faculty at major land-grant universities and USDA facilities ensure that data arising from W-122 collaborative activities will be disseminated to the greatest extent possible among stakeholders and will thus provide maximum benefits to the U.S. public. W-122's efforts and focus are not duplicated in any other regional project.

Impacts of Studying Dietary Bioactive Chemicals. There are a number of positive impacts that will result from this work. First, there will be improved understanding the mechanisms responsible for beneficial and detrimental effects of dietary bioactive chemicals will allow food technologists, production agricultural scientists, health professionals and policy makers to make informed decisions as to when to enhance or reduce the levels of these compounds which ultimately will improve health and reduce risk of the U.S. consumer to a wide variety of chronic diseases. Second, this research will result in the identification of new specialty crops and stimulation of the small farm economy. Third, plant biotechnology firms may he able to engineer increased production of bioactive agents in the patent crop or introduce these agents into different more palatable crops. Fourth, pharmaceutical firms may discover more active derivatives and more effective means of administering the active substances in pure form.

Related, Current and Previous Work

Although the US food supply continues to be among the safest in the world, evidence is increasing that diet is an important factor in our most prevalent chronic diseases. For example, estimates of the impact of diet on human cancers suggest that from 35% to as high as 80% of cancers are related to diet (Weisburger, 2000). Thus, studies in this area hold great promise in reducing human suffering and the great financial burden of associated health care. Investigators in W-122 have made important contributions to our understanding of the significance and modes of action of certain dietary components as toxicants as well as cancer and immune modulators.

Phytoalexins, a complex group, are substances produced by plants in response to various stresses, including attack by a pathogen. Although these substances generally inhibit the growth of the invading organism, they are a source of concern because of their potential toxicity to humans (Beier, 1990). Studies of W-122 committee members have identified the two optical isomers of the polyphenolic substance, catechin, as potent phytoalexins produced by knapweed in response to a fungal pathogen. This work showed that (-)-catechin was toxic to plants, whereas (+)-catechin exhibits antimicrobial activity against a number of organisms. Very importantly, further studies have shown that the readily available, safe and common component of many human foods, (1)-catechin, is a more potent herbicide on broad-leaf plants than the widely used commercial herbicide, 2,4-D, and that it does not affect monocots. Development of this and similar polyphenolic substances may provide at very low cost, a new class of herbicides with very low toxicity to non-target organisms.

Phytoalexins, a complex group, are substances produced by plants in response to various stresses, including attack by a pathogen. Although these substances generally inhibit the growth of the invading organism, they are a source of concern because of their potential toxicity to humans (Beier, 1990). Studies of W-122 committee members have identified the two optical isomers of the polyphenolic substance, catechin, as potent phytoalexins produced by knapweed in response to a fungal pathogen. This work showed that (-)-catechin was toxic to plants, whereas (+)-catechin exhibits antimicrobial activity against a number of organisms. Very importantly, further studies have shown that the readily available, safe and common component of many human foods, (1)-catechin, is a more potent herbicide on broad-leaf plants than the widely used commercial herbicide, 2,4-D, and that it does not affect monocots. Development of this and similar polyphenolic substances may provide at very low cost, a new class of herbicides with very low toxicity to non-target organisms.

Over the last few years the field of immunonutrition has gained increasing importance. Among other dietary compounds, lipids, especially -3 polyunsaturated fatty acids, have been shown to influence the immune response (Grimm et al. 2002). Fatty acids influence inflammatory cell activation processes from signal transduction to protein expression even involving effects at the genomic level. -3 Fatty acid-mediated mechanisms can decrease cytokine-induced adhesion molecule expression and reduce interactions of inflammatory leucocytes with the endothelium. Due to their regulatory impact on different processes of inflammatory and immune cell activation, -3 fatty acids produce positive effects on various states of immune deficiencies and diseases. Dietary fish oil (FO) containing -3 fatty acids is reported to retard the progression of renal disease in patients with the most common glomerulonephritis worldwide. Using an experimental mouse model in which early immunopathological hallmarks are induced by the mycotoxin, vomitoxin (VT), W-122 investigators evaluated the ameliorative effects of FO ingestion on this disease. The results suggested that diets containing FO may impair early immunopathogenesis in VT-induced immune pathology and that this was not totally dependent on the antioxidant activity of FO.

A continuing and growing strength of W-122 is the concentration of several of its members on the characterization of putative cancer protective agents in the diet. Food plants of the Brassica family, including cabbages, kale, broccoli, Brussels sprouts, and cauliflower have received considerable attention for their cancer-preventive activities (Van Poppel, et al. 1999). The results of over 80 epidemiological studies provide strong evidence of the beneficial effects of Brassica-rich diets. The strongest inverse associations between the consumption of Brassica vegetables and cancer risk were observed for lung cancer, stomach cancer, and all cancers taken together. Of the case-control studies, 64% showed an inverse association between consumption of one or more Brassica vegetables and risk of cancer at various sites. The combination of epidemiological and experimental data provide substantial evidence for a cancer preventive effect of a intake of Brassica vegetables.

Collaborative studies have focused the expertise of several W-122 laboratories on a comprehensive evaluation of the modes of action and effectiveness of Brassica indoles as cancer preventive agents. Previous studies from several research groups have shown that the major indole in Brassica plants, I3C, is a strong inhibitor of chemical induced mammary tumors and spontaneous uterine tumors in rodents (Brignall, 2001). Previous studies from W122 investigators and others have shown tumor promoting effects of this indole in liver and other organs. These tumor promoting effects were confirmed recently using a multi-carcinogen model in which rodents were treated with a mixture of carcinogens that produce tumors in mammary gland, liver and other organs (Stoner et al., 2002). Studies at the molecular and cellular level to understand the organ specific effects of the indoles have indicated that I3C is converted to many products on contact with gastric acid following consumption. One of the minor products, ICZ, is a strong activator of the pathway that is activated by the presumed human carcinogen and environmental contaminant, TCDD. Contrary to expectation, low concentrations of the major acid products of I3C show potent estrogenic activity in cultured cells and in trout. The indoles can activate the estrogen response pathway by novel mechanisms that include both direct and indirect activation of the estrogen receptor. These estrogenic effects may contribute to the tumor promoting activities of I3C. Nevertheless, many literature reports claim antiestrogenic activities of the indoles based on an alteration of the ratio of certain estradiol metabolites in women treated with the indoles (Wong et al., 1997). At higher concentrations, the indoles can induce cell cycle arrest and programmed cell death, again by novel mechanisms that target specific cell cycle regulatory factors (CDK6 expression and CDK2 activity). These continuing studies with the Brassica indoles suggest both a good deal of caution in the general use of the substance in cancer prevention, and the promise that a fuller understanding of the complexities of indole action may open new avenues of understanding for organ specific control and prevention of carcinogenesis. For example, recent studies by W122 investigators suggest that the overriding effect of one of the major I3C products is by inhibiting molecular processes central to the later stages of tumor development and metastasis.

Soy products have received considerable attention as possible contributors to the very low cancer rates in reproductive organs in Asian populations. Data from epidemiological reports and laboratory studies have shown that soy isoflavones have multiple biological and pharmacological effects in animals and humans (Ren et al., 2001). Effects include estrogenic and antiestrogenic activities, as well as decreased cell growth and tumor cell death. Consumption of soy products have been associated with reduced incidences of breast and prostate cancers, cardiovascular diseases or osteoporosis. Public interest in the apparent protective effects of soy, combined with the results of some animal studies that seemed to support the role of soy isoflavones in the presumed protective effects of soy in humans, have resulted in a high incidence of self medication with isoflavones in the US. Carefully controlled and executed studies by W122 investigators have established that the soy isoflavones increase estrogen-dependent tumor growth in experimental animals when the isoflavones are administered at the time the carcinogen is administered. Other investigators showed that long term administration of the soy products produced maturing effects in mammary glands that made them less susceptible to tumorigenesis. Collectively, these important studies suggest that whereas consumption of soy products from an early age may be protective against breast cancer, short term, high level consumption of the purified isoflavones in adulthood may promote cancer.

In the culmination of a series of important experiments by W-122 investigators, a large human trial was conducted of the potential cancer protective activities of a chlorophyll product, chlorophyllin (Egner, et al. 2001). Chlorophyllin, a mixture of semisynthetic, water-soluble derivatives of chlorophyll that is used as a food colorant and over-the-counter medicine, has been shown to be an effective inhibitor of aflatoxin hepatocarcinogenesis in animal models that function by blocking carcinogen bioavailability. In a randomized, double-blind, placebo-controlled chemoprevention trial, a large team of investigators that included W-122 members tested whether chlorophyllin could alter the disposition of aflatoxin. The trial was initiated in Qidong, PRC, where residents are exposed to high levels of dietary aflatoxin B1 and the risk of liver cancer is high. This trial aimed to investigate whether dietary chlorophyllin (CHL) treatment may be as effective at reducing human aflatoxin B1 uptake as it was determined to be in trout. The results showed that 100 mg CHL given with each meal over a four-month period reduced urinary AFB1 DNA adducts by an average of 55%. This extraordinary protection was comparable to that seen in trout, and suggests an inexpensive and effective means to reduce AFB1 cancer risk as a major cause of human death in parts of SE Asia and Africa.

Objectives

1. Investigate the cellular and molecular modes of action by which natural bioactive chemicals in food protect against human diseases such as cancer, inflammation and microbial infection.
   
2. Determine cellular effects and molecular mechanisms of natural and induced toxicants in food for human risk assessment and disease prevention.
   
3. Detect and identify new natural or induced bioactive compounds in foods that have beneficial or adverse effects on human health.
   
4. Ascertain how agricultural production and food processing may influence production/stability of natural bioactive chemicals.
   
5. 
   

Methods

Introduction. Through years of interactions, coordination of research efforts and active collaborations, PIs at different stations in the region have developed and maintained expertise and facilities in specific areas which complement and contribute to each other. When these various research capabilities are shared through active collaborations, they greatly enhance and synergize the research productivity of W-122 scientists. Several of the major active and planned collaborative studies among W-122 scientists have been summarized (Table 1), along with the special contributing research capabilities of different Stations (Table 2). All data will be shared among all project members and summarized activities will be accessible to the public via a web site. Objective 1. Investigate the cellular and molecular modes of action by which natural bioactive chemicals in food protect against human diseases such as cancer, inflammation and microbial infection. The following areas are proposed for further research: a) OR will continue investigate the ability of three phytochemical groups - chlorophyll and its derivatives, indole-3-carbinol, and tea polyphenols - to modulate cancer risk. Dose-response, risk-benefit, and combined chemoprevention cancer studies will be carried out in selected animal models (trout liver, stomach, kidney; rat mammary, liver, colon; transplacental mouse lung, liver) in work to be conducted at OSU. Mechanisms of protection (or risk) will be defined through extensive studies at the molecular, cellular, organ and whole-animal level, including gene expression microarray analyses in experimental animals and pharmacokinetic studies with human subjects. CA-Berkeley will collaborate with OR in the exchange of indole-3-carbinol derivatives for study in rodent and human cell culture models and in conducting assays of estrogen responsiveness in human cells. b) UT will assess the potential chemoprotective properties of chlorophyllin and chlorophyll against AFB1-induced toxicity in poultry in collaboration with OR. A variety of endpoints of aflatoxicosis will be measured, such as alanine aminotransferase, aspartate transaminase, lactate dehydrogenase, low-density lipoprotein, and alkaline phosphatase. Since impaired immune surveillance may be also be a mechanism for AFB1 -induced cancer, the potential ameliorative effects on chlorophyllin and chlorophyll on AFB1-induced impairment of cellular and humoral immunity will be addressed in the turkey in collaboration with MI. c) CA-B will use gene expression microarray techniques to determine the kinetics and concentration-dependent effects of the dietary indole, DIM, and related compounds on the expression in cultured breast tumor cells of genes associated with tumor angiogenesis. Promoter analyses will be conducted of key genes implicated in anti-tumor progression activities of indoles to identify regulatory mechanisms for this process. The functional effects of implicated genes on in vitro and in vivo measures of angiogenesis and tumor development, including measures of the immune response will be assessed. CA-B will continue to cooperate with ID on the cancer modulating effects of fermented cabbage, CA-USDA on the estrogenic activities of potato alkaloids, and MI on the immune/inflammatory effects of the Brassica indoles. d) MI will continue to evaluate the effects of omega-3 fatty acids as well as three herbal chemicals , apigenin (chamomile), ginsenoside (ginseng), and parthonelide (feverfew) on models for autoimmune and inflammatory diseases using molecular endpoints related to signaling (mitogen activated protein kinases) and gene expression of inflammation/immune mediators. Collaborative research will be conducted on effects of dietary indoles (CA-B), dietary estrogens (IL), antioxidants (UT), plant-derived sphingosine pathway inhibitors (GA-USDA) on signaling and gene expression associated with inflammation/immune function. e) CA-USDA will (a) determine relative antimicrobial activities of structurally different phytochemicals (structure-function relationships); (b) assess synergism of mixture of two or more compounds; and )) measure retention of bactericidal activity when active compounds are incorporated into foods. Other bioassays will be provided by collaborators antitumor effects OR anti-toxic effects (UT), cell-signalling effects (GA-USDA), immune effects (MI) and estrogenic activities (CA-B). f) ID will use an established rodent colon tumor model in conjunction with CA-B and OR to study the effects of bioactive indoles on the formation of preneoplastic lesions induced by a chemical carcinogen in the colon. Protein alterations which may be important in the development of cancer in colon epithelial cells will be examined. Objective 2. Determine cellular effects and molecular mechanisms of natural and induced toxicants in food for human risk assessment and disease prevention. The following issues are proposed for further research: a) OR will investigate the mechanisms through which selected food-borne toxicants (aflatoxins; dibenzo[a,l]pyrene; heterocyclic amine cooked meat mutagens) induce cancer. This will be done by characterizing the types of DNA damage that occur, the specific gene mutations that may result, and the alterations in the function of genes that regulate cell proliferation and apoptosis in target organs in lower and higher vertebrate models. b) UT will study the effect of AFB1 on tumor suppressor gene p53 in cultured human lung cells. Effects on apoptosis, and expression of activated p53 protein will be measured. c) MI will elucidate molecular mechanisms by which trichothecene mycotoxins suppress immune function by focusing on the regulation of genes associated with cell death (apoptosis) in leukocytes in primary cell cultures from mouse and human blood as well as mouse models. Structure/function effects of related 190 naturally-occurring trichothecene will be addressed in this research. MI will collaborate with CA-B, IL, GA-USDA in the of the effects dietary indoles, phytoestrogens and sphingosine pathway modulators on apoptosis in mouse and human lymphocytes. d) AZ will study mechanisms of PAH-induced tumor formation. Research methods will include analysis of changes in gene expression of cell cycle checkpoints including promoter studies, ribonuclease protection assay, electrophoretic mobility shift assay, western blotting, and flow cytometry, AZ will exchange materials and collaborate on bioassays with other labs investigating genotoxicity induced by PAHs (OR, IL), carcinogencity of toxins (CA-D, OR, UT), DNA-crosslinking (UT), and molecular mechanisms of anti-carcinogenesis (CO, OR). e) IL will utilize the athymic mouse human tumor implant model to evaluate the interaction of dietary estrogens and therapeutic drugs. This model will generate dose- response data on the epigenetic carcinogenic effects of phytoestrogens in susceptible human populations. Currently this model is the most widely used model to evaluate the effectiveness of anti-estrogen therapy. Objective 3. Detect and identify new natural or induced bioactive compounds in foods that have beneficial or adverse effects on human health. Certain of the bioassays employed in Objectives 1 and 2 have been refined sufficiently to be applied to the identication novel natural bioactive chemicals. The following issues are proposed for further research: a) CO will isolate novel beneficial substances which are ordinarily produced in small amounts and difficult to isolate and identify using a novel system involving elicitation in hairy roots, grown in culture. The production of these substances will be enhanced by the use of elicitors (jasmonic or salicylic acid, pathogen lipopolysaccharides, etc.) which have been shown to induce increased secondary metabolite production. After elicitation and growth, bioactive compounds will be isolated from the medium using bioassay-directed fractionation. CO will specifically employ an antimicrobial assay. Other bioassays of these novel compounds will be provided by collaborators at antitumor effects (OR), anti-toxic effects (UT), cell-signalling effects (GA-USDA) and anti-inflammatory and immune effects (MI). b) GA-USDA will identify novel bioactive plant products that can modulate sphingolipid biosynthesis and calcium regulation and thus interfere with cell homeostasis. The mechanism by which these plant products act at the cellular level will be further characterized to determine the potential beneficial or adverse effects of these chemicals using animal models for (I) toxicity at GA-USDA, (ii) carcinogenesis at OR, AZ and ID, (iii) inflammation and immunity at MI. c) UT will study a variety of alkaloids extracted and purified from western plants DNA on cross-link formation in synthetic oligonucleotides. d) ID and GA-USDA will assist in the characterization of newly isolated natural chemical products to determine the existence and levels of bioactive compounds such as antioxidants, phenolics, flavones, etc. of interest and develop analytical methods for quantification. HPLC, GC/MS, LC/MS, MS/MS will be available for the analytical projects. OBJECTIVE 4. Ascertain how agricultural production and food processing may influence production/stability of natural bioactive chemicals. The following issues are proposed for further research: a) CA-D is currently studying the influence of pesticides on mycotoxin production. The effects of conventional, IPM, and/or organic practices on output of natural toxins will be investigated by coupling In vitro laboratory studies with in vivo field studies. This will be expanded include other natural toxins and novel endogenous bioactive chemicals. b) CA-USDA will investigate the effects of food processing and genetic modification on bactericidal activity of active natural chemicals. Other bioassays will be provided by collaborators at OR, UT (antitumor), GA-USDA (cell-signalling) and MI (inflammatory/immune).

Measurement of Progress and Results

Outputs

• Consumer information on the beneficial and adverse impacts of bioactive, dietary chemicals on human health and chronic disease.
• Improved recommendations and guidelines for use of bioactive dietary compounds in herbal supplements and functional foods.
• Improved hazard and risk assessment data are of dietary toxicants for policy makers.
• Identification of novel value added crops and foods that can be exploited by farmers and processors, respectively.
• Creation of new antimicrobial foods to protect both the food and consumers against human pathogens.
• <p>Dissemination of data in scientific meetings and peer-reviewed journals.</p> <p>Purified natural chemicals, complex mixtures (extracts) with biological activity, biological reagents such as antibodies, cell lines, RNA from treated cells and laboratory animals will be made available for further study to other W-122 researchers.</p>

Outcomes or Projected Impacts

• Reducing cancer incidence and increasing safety by characterizing the beneficial and adverse effects of putative cancer protective substances from food. This information will assist the National Cancer Institute in selecting agents for clinical intervention trials, and the US-FDA and other regulatory agencies to regulate and properly evaluate the safety and effectiveness of certain widely used food supplements.
• Improving human health by understanding and exploiting the role of dietary components on immune function. Dietary immunomodulation holds great promise for decreasing human morbidity and mortality from many causes.
• Benefitting growers, the American agrochemical industry, and improving the safety of the food supply by developing plant-food derived substances as safe, effective and economical antimicrobial and herbicidal agents.
• Benefitting the American pharmaceutical industry by developing Brassica indoles and isoflavones as model gene targeting reagents for use in cancer therapy and prevention. These and other food constituents hold great promises as models for the development of safe, selective, and effective cancer protective.

Milestones

(0):edule of proposed studies is contained in the attached Milestone.ppt

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

New information and conceptual insights resulting from this research will be communicated at several levels. The full details of the research will be published for world wide distribution in peer-reviewed scientific journals. Findings will be presented as updates and extensions to teaching modules for students at all levels of advancement from primary schooling to graduate and post graduate education. Findings that are considered important for public distribution will be placed into a format that is suitable for dissemination through the media. When our level of understanding of new findings and their significance so dictate, the information can become the focus of workshops and training sessions for Cooperative Extension Specialists. W-122 is in the process of establishing a website for rapid distribution of important information and for communication of constituents with W-122 members.

Information resulting from investigations of W-122 members is important to several traditionally underserved groups and is made readily available to them by diverse means. For example, in areas such as the San Francisco Bay Area, the incidence of breast cancer is the highest in the world and the level of concern of women in this area is intense. W-122 investigators are working closely with community information organizations to provide perspectives on the possible role of diet in this disease. Contributing Experiment Stations have developed effective means of communicating with poor and minority populations through cooperation with rural churches, intercity food banks, and food stamp advisory organizations. Undergraduate minority student training programs are also effective means of providing information to consumers and potential future community leaders. Several Experiment Stations, also have well developed lines of communication with associations of growers for which W-122 findings will be useful.

Organization/Governance

The Technical Committee consists of the Administrative Advisor, representatives of the various Research Divisions of the U.S. Department of Agriculture, the CSREES-USDA, and a designated representative from each participating experiment station. An Executive Committee will consist of the chairman, vice-chairman, and secretary. Each year a new secretary will be elected and the officers advanced, the secretary becoming vice-chairman, the vice-chairman becoming chairman. Thus, each person elected shall serve as secretary, vice-chairman, and chairman during a consecutive three year term. The Executive Committee will conduct annual business meetings as called by the Administrative Advisor, and will be empowered to act for the Technical Committee between annual meetings. At the annual meetings, research will be reviewed and cooperative efforts and research priorities within the objectives of the regional research proposal will be established.

Literature Cited

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Brignall, M, (2001) Prevention and treatment of cancer with indole-3-carbinol. Altern Med Rev 6:580-9.

Egner PA, Wang JB, Zhu YR, Zhang BC, Wu Y, Zhang QN, Qian GS, Kuang SY, Gange SJ, Jacobson LP, Helzlsouer KJ, Bailey GS, Groopman JD, Kensler TW., (2001) Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer. Proc Natl Acad Sci U S A 98:14601-6.

Gaffield W, Keeler RF. (1996) Induction of terata in hamsters by solanidane alkaloids derived from Solanum tuberosum. Chem Res Toxicol. 9(2):426-33.

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Land Grant Participating States/Institutions

AZ, CA, CO, HI, IL, MD, MI, OR, TX, UT

Non Land Grant Participating States/Institutions

USDA-ARS/Georgia, USDA/ARS-California