Duration: 10/01/2007 to 09/30/2012

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Overview: This application represents a renewal of a productive regional project that has been in existence since 1971. With comprehensive participation, the overall goal of W-1122 is to improve food safety and human health worldwide. Research supported by W-1122 addresses natural foodborne toxicants, cancer chemopreventives, phytoestrogens, immune modulators and antimicrobials. A particular strength is our dual focus on understanding the molecular basis underlying the impact of dietary chemicals on human health, and applying this knowledge to real-world problems in clinical trials, and intervention studies. These efforts cover a spectrum of cutting-edge scientific approaches that include molecular signaling, genomics, tumor formation, and molecular cancer dosimetry and biomarkers. While some objectives of this renewal application continue W-1122's established emphasis on the role of natural dietary compounds in human health, many new avenues of research are proposed, reflecting a continual evolution and refinement in the state-of-the-art. W-1122 has been highly successful in meeting its objectives, as measured by its numerous collaborative efforts leading to discovery of adverse and beneficial effects of natural food compounds.

Issues related to food safety and to diet and health impact many stakeholders. Understanding the complex relationship between dietary chemicals and human health remains a paramount concern to our primary stakeholders who include consumers, agricultural producers, food processors, health professionals, and policy makers charged with maintaining a safe and nutritious food supply. Food-borne bioactive chemicals are defined in this proposal as naturally occurring and processing-induced substances that exert beneficial or undesirable effects when consumed. This multi-state project does not focus on synthetic agrochemicals and environmental pollutants.

A primary issue is the potential of dietary chemicals to induce or exacerbate disease. For example, certain genotoxic fungal toxins can induce cancer by direct DNA damage or in an epigenetic fashion by tumor promotion. Indeed, excluding smoking, diet-related factors 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). The public increasingly recognizes that people differ very greatly in their response to chemical carcinogens ("Environment and Cancer: The Links Are Elusive", NY Times, Dec. 13, 2005). 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 stakeholder 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, W-1122 participants have shown that chlorophyll and its derivatives can function as anticarcinogens and block the action of food-borne genotoxic chemicals such as aflatoxin and heterocyclic amines. Bioactive dietary chemicals can affect other chronic diseases. Omega-3 fatty acids which are produced by plants and algal fermentations 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 stakeholder concern is the often baffling complexity associated with separating the positive and negative attributes of a dietary chemical. Often possible benefits are perceived as "hypothetical and elusive" ("Which of These Foods Will Stop Cancer? (Not So Fast)" NY Times, G. Kolata, September 27, 2005.). Notably, dosage and timing of exposure can determine whether these bioactive compounds are beneficial or detrimental. W-1122 researchers have a considerable history in this risk-benefit field. 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 represent an extraordinarily diverse group with respect to chemical structure and biological activities. The numerous kinds of dietary toxicants, including the structurally diverse metabolites of molds and bacteria, illustrate this diversity. Aflatoxin B1 is a potent carcinogenic mycotoxin often found in U.S. foods such as corn, peanuts and milk. Recent studies indicate that contamination of the U.S. corn supply by another mycotoxin, fumonisin, may represent an even greater hazard due to its carcinogenic and teratogenic properties. Wide-spread Fusarium mold contamination of wheat and barley and resultant deoxynivalenol (DON; or "vomitoxin") contamination often occur prompting food/cereal companies and the brewing industry to stop purchasing these commodities from the U.S. farmers. 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 of further significance because of their transfer from feed to animal tissues used for food. Research by W-1122 members addresses mechansistic risk assessment of these mycotoxins that is being used by regulatory agencies in the U.S. and through the world. Other research deals with strategies for mitigating human health impacts of mycotoxins. In addition to the mycotoxins, the food supply can be contaminated with numerous other substances with significant toxicity, including bacterial toxins, industrial pollutants and carcinogens produced from cooking of meat, such as heterocyclic amines and polycyclic aromatic hydrocarbons.

Brassica indoles and chlorophyll 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, and have become the preferred treatment for recurrent laryngeal papillomas in humans. One of these indole products, DIM, is undergoing clinical trials as a treatment against cancers of the breast, prostate and colon. Chlorophyll and its derivatives have the potential to reduce cancer risk by sequestering active carcinogens in the gastrointestinal tract which reduced carcinogen exposure. Results of a human intervention trial in China showed that administration to humans of the chlorophyll derivative, chlorophyllin, could markedly reduce the level of DNA damage caused by aflatoxin B1 (Egner, et al., 2001) Risk of liver, colon, stomach, breast, prostate, and lung cancer, which include the leading causes of cancer death in the US and the world, may be significantly reduced by appropriate daily intake of simple, safe, inexpensive indole and chlorophyll derivatives. Whether synthetic analogues of the Brassica indoles will have improved protective activities, is a subject of our further investigations. Continuing studies suggest that native chlorophylls in green vegetables have protective effects that are similar to chlorophyllin against carcinogen absorption from the gut. 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 and certain herbal supplements, 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 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. Studies from the W-1122 group have elucidated the molecular signaling pathway by which omega-3 fatty acids can inhibit an autoimmune kidney disease. Clearly, failure to exploit the potential for bioactive dietary chemicals to ameliorate such diseases would have far-reaching social, economic, and health impacts.

In some cases, bioactive dietary chemicals have paradoxical effects. For example, the phytoestrogen, genistein, occurs in significant amounts in soy products and is widely used as a supplement for various maladies of possibly hormonal origin. Genistein has been shown to reduce the number of DMBA-induced mammary tumors when administered early (prior to puberty) to rodents. The likely mechanism responsible is an estrogenic effect of genistein, which causes an enhancement of mammary gland differentiation and a reduction in mammary cell proliferation that ultimately reduces the amount of DNA damage from the DMBA. This presents a significant paradox because when genistein is administered to rodents with estrogen responsive tumors, the estrogenic effect produces an enhancement of tumor growth. W-1122 researchers have also shown that Brassica indole, 13C, is a tumor promoter in a multisite cancer model. Further studies of the beneficial and potentially hazardous effects of genistein and other soy products, as well as Brassica indoles, are being conducted by W-1122 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. 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. 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 have 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 Johns Wort to act in an additive manner with anti-depressant drugs and the ability of extracts of many herbal products to activate enzymes involved in drug metabolism (Jeuken et al., 2003).

The focus of W-1122 is primarily on plant products, compounds derived from the growth of microbes on foodstuffs, and substances created (induced) during food processing. An additional category of induced bioactive components is 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 the incidence of other chronic diseases. 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 Natural Dietary Chemicals: The research proposed herein exploits recent technical and conceptual advances in biomedicine. New knowledge of the regulatory pathways for cell cycle, cell differentiation and programmed cell death and their relationship to disease allows the identification and exploration of cellular control mechanisms. W-1122 members have established an international reputation in research into mechanisms and hazards of dietary toxicants as well as exploitation of beneficial natural dietary compounds to improve human health. We have extensive experience in a variety of model systems (human and animal cell culture, transgenic and knockout mice, mouse transplacental transport, rats, poultry, plant, rainbow trout, human subjects) with which to pursue this work. Using these models, members have established the benefits of chlorophylls, indoles, anti-oxidants, omega-3 fatty acids, various antimicrobials and plant growth factors. These models have also been used by W-1122 investigators to identify potential adverse effects of mycotoxins, indole-3-carbinol, phytoestrogens, and induced toxicants, demonstrating that the effects of natural dietary compounds are sometimes double-edged, and that careful selection of recipient populations or individuals is essential to optimize benefit over risk.

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. 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, chemistry, molecular biology, genomics, nutrition, food science and risk assessment) as well as geographic diversity due to the wide range of food crops that may be involved. W-1122 members have collaboratively studied bioactive dietary chemicals and 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 facilitate goal achievement and limit duplication of effort among member's respective units. Their positions as faculty at major land-grant universities and USDA facilities ensure that data arising from W-1122 collaborative activities will be disseminated to the greatest extent possible among stakeholders and will thus provide maximum benefits to the U.S. public. W-1122'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 and foremost, this research will directly result in an improved understanding of the mechanisms responsible for the beneficial and detrimental effects of dietary bioactive chemicals. This knowledge will help form the basis for an informed evolution of dietary habits to enhance or reduce the levels of these compounds to improve health. Second, this research will result in the identification of specialty crops and stimulation of the small farm economy. Third, biotechnology firms may be 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 reasonably safe, evidence is increasing that diet is an important factor in our most prevalent chronic diseases. Estimates suggest that diet is responsible for between 35-80% of human cancer incidence (Weisburger, 2000). Furthermore, genetics appear to play a smaller role in human cancer than once thought. Indeed, only a few cancers - breast, prostate and colorectal - appear to have an appreciable genetic component (Lichetenstein et al., 2000). Thus, identifying potential adverse and beneficial dietary components holds great promise in reducing human suffering and the great financial burden of associated health care. Collaborative research supported by the W-1122 multi-state project has 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. A continuing strength of W-1122 is the focus on molecular modes of action of dietary carcinogens and toxins as well as natural and synthetic cancer chemopreventive agents. Research particularly focuses on phase I and II biotransformation pathways that underlie toxicity, cancer, and cancer chemoprevention, as well as pathways and transcription factors important in cell division, tumor suppression, and apoptosis.

The results of over 80 epidemiological studies provide strong evidence of the beneficial effects of Brassica-rich diets, and that consumption of cruciferous vegetables protects against cancer more effectively than the total intake of fruits and vegetables (Keck, 2004). Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) are two such potential chemopreventive agents from Brassica. In our continuing studies of the efficacy and modes of action DIM, we have uncovered several functions and novel mechanisms of action relevant to multi-organ cancer prevention and therapy. These potentially protective processes include apoptosis, estrogen receptor-independent G1 cell cycle arrest, up-regulation of protective p21 expression in human breast cancer cells, induction of transforming growth factor (TGF)-mediated cell cycle arrest in endometrial tumor cells, potent antiproliferative and antiandrogenic properties in androgen dependent human prostate cancer cells, immune enhancement, and induction of lymphocyte proliferation and potentiation of gastric clearance of reovirus. DIM also inhibits tumorigenesis in human breast tumor xenografts in rodents by a mechanism that involves the inhibition of tumor angiogenesis. Taken together, these studies have identified several complementary effects and modes of action of DIM that may be exploited in the development of this natural substance as a cancer chemopreventive or therapeutic agent (Le, et al., 2003). This work has provided the impetus for human clinical trials for several types of cancer. At the same time, W-1122 researchers have determined that the principal mode for tumor enhancement by indoles in animals lies in their potential for estrogenic effects in the liver (Tilton et al., 2005). Exploration of such effects in humans will be essential for establishing human risk, if any, and for identifying individuals most likely to benefit from indole intervention. This continuing research with Brassica indoles hold have 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.

W-1122 researchers have pioneered rainbow trout as a model to establish risk to dietary carcinogens and to identify potential chemopreventives. The model solves a major logistical difficulty inherent in using large numbers of rodents to predict an incidence of 10-6 above background, with the advantages of low spontaneous tumor incidence, high sensitivity to a range of carcinogens, low animal purchase and per diem costs, established record of husbandry, exposure protocols, dose-response and pathology and mechanisms of metabolism, DNA adduction, oncogene activation and pathology similar to mammalian models. A recent 42,000 trout ultra-low dose tumor study utilizing dibenzo[a,l]pyrene (DBP) established the dose-response to 1 additional cancer in 5,000 animals, a 50-fold improvement over rodent models. A significant negative departure from the EPA conservative linear model was observed with decreasing DBP dose, leading to a predicted dose of DBP resulting in 1 additional cancer in 106 1000-times higher than predicted from linear extrapolation. If applicable to other genotoxic carcinogens, the implications of these findings in risk assessment are profound. In this application, we propose to use this model to determine the dose-response curve at low doses with AFB1 that should shed new light on the risk posed by this ubiquitous carcinogen. Another in vivo model pioneered by W-1122 researchers is a unique mouse model of transplacental induction of the most common type of childhood cancer in humans, lymphoma/leukemias, to identify cancer chemopreventives (Yu, et al., 2006). Agents currently under study are I3C from cruciferous vegetables, polyphenols in teas and chlorophylls from green and leafy vegetables.

Dietary phytoestrogens such as soy isoflavones are aggressively marketed to women as a natural and perceived safe alternative to hormone replacement therapy. While in most cases soy is a healthy food, it has the potential to be a risk factor for metastatic breast cancer when consumed in pure or enriched forms. Because metastasis is the main cause of mortality in breast cancer patients, the safety of dietary phytoestrogens is an important area of food safety research. Appropriately, W-1122 investigators will continue to evaluate the effect of dietary phytoestrogens on breast cancer rates in a preclinical animal model. Evaluation of metastasis will involve the injection of human breast cancer cells into mice and following tumor occurrence in lung, kidney, liver and bone. The effect of dietary phytoestrogens on the number and size of these tumors using an in vivo laboratory model will be evaluated.

The ability of naturally-occurring chemicals present in a variety of food products to inhibit growth and proliferation of a variety of human cancer cells via their ability to interact with nuclear receptors (predominantly the estrogen and Ah receptors) suggests the possibility of using whole foods (vegetables and dietary herbal products), food extracts and synthetic chemicals based on the structure of natural chemicals to stop or slow cancer growth. W-1122 investigators will continue to carry out nuclear receptor bioassay-based fractionation approaches directed toward the isolation, identification and biochemical characterization of the responsible chemicals present in these foods. These studies will allow the development of these new naturally occurring dietary components and/or synthetic derivatives of these chemicals as chemotherapeutic agents.

Immunonutrition is an emerging emphasis area in food safety research. W-1122 researchers identified natural immune system modulators that can potentially protect against a range of disease processes from viral infection to cancer. For example, omega-3 fatty acid consumption by mice causes macrophages of the innate immune system to be reprogrammed. As a result, induction of inflammatory gene expression by toxins and stress is suppressed thereby leading to decreased disease. To translate this into benefits for humans, optimal omega-3 fatty acid doses and mechanisms must be first identified. Collaborative work between MI and UCB investigators found that dietary indole consumption markedly stimulates the immune systems and decreases the capacity of respiratory enteric orphan virus to cause gastrointestinal infection. The mechanisms for this immune stimulation and doses necessary for therapeutic action must be determined.

As exemplified by the Escherichia coli O157:H7 outbreak from bagged spinach in September 2006, there is an alarming increase in the number of foodborne illness outbreaks. The increasing antibiotic resistance of some pathogens associated with foodborne illness is another concern. Therefore, developing new types of effective and non-toxic antimicrobial compounds is an important endeavour. Plant-derived compounds, some of which are root exudates, volatile organic chemicals, or plant extracts are potentially useful sources of antimicrobial compounds against foodborne pathogens including Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, Bacillus cereus, and Clostridium perfringens (Friedman et al., 2002). Some may be used as value-added natural pesticides to improve food safety by reducing the application of synthetic chemicals (Bais et al., 2006). How best to use these naturally occurring, food-compatible and safe compounds to protect foods and feeds against pathogenic bacteria is an emphasis of the proposed work. Basic and applied knowledge of plant antimicrobials is crucial to addressing known and unanticipated problems in microbial food safety that may occur due to changes in food production processes, adaptation of pathogens in production systems, and in bioterrorism. Some of these beneficial compounds are root exudates.

Fumonisin is a neurotoxic mycotoxin postulated to also be responsible for in utero neural tube defects (NTD) in humans. W-1122 researchers completed a five year collaborative study with the Instituto de Nutricion de Centro America y Panama that measured the levels of fumonisins in maize grown in the highlands and lowlands of Guatemala (Riley and Pestka, 2006). The data were used to develop an exposure assessment for women in Guatemala. The results showed that a significant number of women in Guatemala are exposed to levels of fumonisin that exceed the tolerable daily intake recommended by the WHO/FAO. Based on the exposure assessment, recommendations for minimizing fumonisin exposure were provided to the Consejo Nacional de Ciencia y Tecnologia in September of 2005. A consumer education program has been funded in Guatemala for the purpose of ensuring that consumers are aware of steps in traditional processing that should be taken to improve the safety of maize products. Future studies are needed to determine the role of fumonisin exposure and folate deficiency in NTD incidence in Guatemala.

Related regional projects. That the mission and objectives of W-1122 is unique was confirmed by searching National MRF portfolio on the NIMSS site performed August 2007. Multistate project NC1025 Mycotoxins: Biosecurity and Food Safety (start: Sep 2005) is focused on mycotoxins in cereal grains: risk assessment in human and animal health, developing new detection techniques, management and prevention strategies, and biosynthesis and ecology of mycotoxin-producing fungi. Our project objectives have no no overlap with NC1025.
Project NC1167 (N-3 Polyunsaturated Fatty Acids and Human Health and Disease) is focused on determining the effectiveness of agricultural-based n-3 PUFA as compared to marine-based n-3 PUFA to promote health and prevent disease and identify agricultural and marine sources of n-3 PUFA to meet new dietary guidelines. The work on n-3 PUFAs in proposed W-1122 focuses on molecular mechanisms for anti-inflammatory effects and hence does not duplicate the efforts of NC1167 but rather is complementary. To ensure complementation, MI W-1122 representative discusses n-3 efforts regularly with department colleague Dr. Kate Claycombe, the MI-NC1167 representative. The uniqueness of W-1122 was confirmed in an Assisted CRIS search of regional projects using key words from this proposal. Thus, the scope of the W-1122 is unique to regional projects because it emphasizes improving human health and to understand molecular mechanisms of carcinogenesis and chemopreventives in human disease prevention.

Objectives

1. Consumption of food-borne bioactive compounds can protect against human diseases such as cancer, inflammation, birth defects, and microbial infection. We will determine the mechanisms by which selected compounds exert their protective action.
   
2. Food-borne toxins and carcinogens are present per se or are induced by processing, preparation, and other post-harvest steps. We will identify mechanisms of action and develop biomarkers of natural and induced toxicants in food for human risk assessment and disease prevention.
   
3. Selected classes of bioactive compounds show potential for beneficial or adverse effects on human health. We will discover bioactive compounds that have beneficial or adverse effects on human health.
   
4. Modifying foods is an increasingly important strategy to improve nutrition and safety. Therefore, we will improve food safety by developing approaches to increase beneficial or decrease adverse effects of bioactive food constituents and microbial contaminants.
   

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-1122 scientists. Several of the major active and planned collaborative studies among W-1122 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 W-1122 web site. Objective 1. Epidemiological and some laboratory data strongly indicate that certain food-borne bioactive compounds protect against human diseases such as cancer, inflammation, birth defects, and microbial infection. We will determine the mechanisms by which selected compounds exert their protective action. The following areas are proposed for further research: a) OR will continue to employ a comparative approach in elucidation of the mechanisms of action, at the molecular and cellular level, of three major classes of phytochemicals, indole-3-carbinol from cruciferous vegetables, chlorophylls from green and leafy vegetables and green tea. The primary target will continue to be cancer. Basic mechanisms of cancer chemoprevention are studied in trout and mice and then small clinical trials (mostly focused on bioavailability, metabolism, etc.) will be performed. OR will continue to collaborate with CA-Berkeley, specifically in the synthesis, characterization and mechanism of action of indole-3-carbinol and derivatives. Additionally, OR will collaborate with CA-D to evaluate the effects of these phytochemicals on nuclear receptor function, specifically that of the estrogen, androgen and Ah receptor signaling pathways. b) Antioxidants have been shown to reduce bioavailability, increase excretion, and reduce residues of AFB1 in turkey meat products, and therefore is a potential strategy to improve food safety. In collaboration with OR, UT will assess the potential chemoprotective properties of antioxidants against AFB1-induced toxicity in poultry as well as on residues of mycotoxins such as AFB1 in commercially-important tissues, such as breast meat and fat. Determination of residual antioxidants will also be determined to insure that the levels fall within acceptable limits set by the US FDA. Phase I and phase II enzymes important in AFB1 bioactivation and detoxification, such as cytochromes P450 (CYP) and glutathione S-transferase (GST) will be characterized for their role cancer susceptibility and resistance. c) CA-B will test the important hypothesis that DIM and other cancer protective phytochemicals, including resveratrol and curcumin, function as pro-oxidants to induce programmed cell death and to decrease tumor metastasis. These scientists will characterize the anticancer mechanism of action of DIM by determining its interactions with mitochondrial F1F0-ATP synthase and functional consequences on cellular levels of O2 and reactive oxygen species (ROS) in breast cancer cells that express a mutant form of F1F0-ATP synthase that is insensitive to DIM compared to wild-type cells. The role of activation of a specific prolyl hydroxylase enzyme in the mechanism of DIM mediated degradation of the major hypoxia response regulatory protein, HIF-1a, in hypoxic breast tumor cells also will be determined. In collaboration with MI, CA-B will examine the mechanism by which DIM can augment the immune response and determine the functional significance to this effect in prevention of cancer and viral diseases. These investigators will test the hypothesis that DIM augments the immune response by inducing the liberation of ROS and resultant activation of immune responses. CA-B plans to continue with several active collaborations with colleagues in the W1122 group. Studies with OR will continue to examine the cancer modulating and possible toxic effects of Brassica indoles in the trout cancer model and in a rodent model of prenatal development. Studies with CA-ARS will explore the antimicrobial and immune enhancing effects of DIM in rodent models of infection. MI will collaborate with CA-B in relating DIM consumption to antiviral activity using mouse models of respiratory infection (reovirus, influenza) and enteric tract infection (reovirus, norovirus). These studies along with CA-B findings will be used as a basis for uncovering the molecular mode of action for enhanced immunity with the goal of eventual application to immune enhancement in human subjects. d) IL will evaluate protective/detrimental effects of phytoestrogens using a combination of in vivo and in vitro breast cancer models. In addition, IL will collaborate with CA-D to evaluate the anticarcinogenic effects of crude extracts and natural products isolated from a variety of herbal supplements and foods. In addition, other natural products and resulting synthetic derivatives of these products that are active in nuclear receptor bioassay systems will be evaluated for their ability to affect growth and proliferation in vivo and in vitro of breast cancer models. e) CA-ARS will determine antimicrobial activities of different classes of naturally-occurring, plant-derived, food-compatible antimicrobial compounds against non-resistant and antibiotic-resistant foodborne pathogens including Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens. The antimicrobial effectiveness of plant compounds incorporated into edible fruit and vegetable films, and applications of the most active compounds and plant extracts in different food categories including fruits, vegetables, fruit and vegetable juices, meat, and poultry products will be determined using LC50 values. Toxin inactivation will be determined by ELISA, tissue cultures, and LD50 determination in mice to determine possible synergistic or other addition effects. f) MI and GA-ARS will collaborate to determine the effects of omega-3 fatty acid consumption on immune-mediated diseases including glomerulonephritis, asthma and food allergy, as well as how it suppresses toxic responses to fungal,plant and bacterial ribotoxins. g) GA-ARS, CA-ARS, OR will work together to conduct animal and field studies to determine the role of the mycotoxin fumonisin (FB) exposure and folate intake on development of neural tube defects in humans. Dose-response, effect of exposure time, and role of placenta for neural tube defect (NTD) and fetotoxicity under conditions which disrupt maternal sphingolipid biosynthesis will be determined in LM/Bc mice. Field studies in Guatemala will continue to genetically screen NTD patients and their families in Guatemala, and to estimate FB exposure at the time of conception in NTD affected populations. h) MD will investigate anticarcinogenic properties of anthocyanins and flavonoids in colon cancer model systems (in vitro and in vivo). CA-B, UT and OR will cooperate in identifying mechanisms for these effects. i) HI will study anti-obesity, anti-diabetic effects of natural products in animal and cell culture models and cooperate with CA-B and MI to identify signal transduction mechanisms. Objective 2. Food-borne toxins and carcinogens are present per se or are induced by processing, preparation, and other post-harvest steps. We will identify mechanisms of action and develop biomarkers of natural and induced toxicants in food for human risk assessment and disease prevention. The following studies are proposed: a) OR will continue to utilize the rainbow trout to conduct large scale cancer studies, not practical in rodent models, to enhance human risk assessment for food carcinogens such as dibenzo[a,l]pyrene (from cooked meats) and AFB1. The applicability of biomarkers, among which will be patterns of gene dysregulation as revealed by microarrays, will be determined. OR will also utilize 51,600 trout to determine the dose of AFB1 resulting in 1 additional liver tumor in 1,000 animals; determine if the incidence at low dose progressively departs from linearity. Animals will be sub-sampled at each AFB1 dose group to measure liver AFB1-DNA adduction, cell proliferation, apoptosis and gene expression by microarray. b) Researchers from UT have recently cloned and expressed CYP1A5 from poultry that has high activity toward AFB1 bioactivation. This gene bears substantial homology to to human CYP1A2 which bioactivates many dietary carcinogens, such as AFB1. Thus, the turkey homologue can be considered as a model for a human CYP gene associated with cancer risk, and studies here can provide information on the molecular basis of cancer susceptibility in people. In collaboration with OR, this gene will be mapped with the goal of establishing molecular determinants in conserved motifs that underlie hypersusceptibility toward this potent hepatocarcinogen. In addition, the molecular mechanism of regulation of expression of CYP1A5 and the implications on AFB1 bioactivation will be carried out in collaboration with CA-D. c) MI will collaborate with GA-ARS by providing tissue and samples from ongoing acute and short-term chronic DON exposure studies for identification of serum-based metabolomic and proteomic markers of toxicity. To enhance MI will share new data relating increased gene expression of suppressor of cytokines(SOCs) proteins to suppressed action of growth hormone. These data will facilitate accurate prediction of adverse human health effects and enable science-based risk assessment for this mycotoxin. d) GA-ARS will collaborate with CO to determine the dose response and tissue specificity for fumonisin-induced elevation in sphingoid base 1-phosphates (S 1-P) and toxicity in mammalian and plant models and evaluate the use of elevated S 1-P as a biomarker of exposure and disease susceptibility using animal and plant bioassays. e) MD has demonstrated age-related adverse effects observed with interaction between the phytoestrogens curcumin and soy isoflavones and the colon carcinogen azoxymethane, where chemoprevention was observed in young and old, but not mature rats. MD will extend these studies in two directions. Firstly, we will utilize the same model (azoxymethane in different aged animals) to determine structural or molecular mechanisms that may be responsible for the age-related differences in response to dietary interventions. Secondly, as azoxymethane is metabolized by CYP2E1, MD in collaboration with UT will assess the role of CYP2E1 in this age-related response. To confirm whether modulations of the metabolic enzymes are involved in the age-related differences, specific enzyme inhibitors and other drugs metabolized by this pathway may be used. Objective 3. Selected classes of bioactive compounds show potential for beneficial or adverse effects on human health. We will discover bioactive compounds that have beneficial or adverse effects on human health. a) To determine whether anthocyanin and flavonoid levels of teas, wines, and fruit and vegetable juice predict biological potencies, CA-ARS will compare antibiotic activities of flavonoids (e.g. epigallocatgechin-3-gallate present in green tea and theaflavin-3-gallate present in black teas) to corresponding activities of tea infusions with known composition. b) GA-ARS will determine chemicals responsible for the virulence of Fusarium verticillioides in corn-seedling disease and on root development and leaf lesions. The ability of fumonisin producing and non-producing strains to cause maize seedling disease, as well as if other chemicals produced by F. verticillioides contribute to virulence will be determined. This information will provide valuable insights for marker-assisted selection for maize varieties resistant to fungal infection and possibly provide strategies for reducing the accumulation of the toxins responsible for animal and human toxicity. c) Using a variety of analytical and molecular biology techniques, CO will identify new antimicrobial and anti-infective compounds, phytotoxins, and other compounds involved in chemical/ ecological interactions in plants. The compounds will be investigated for their agriculture-related and ecologically benign applications (such as natural pesticides) and will provide a better appreciation of the value of plants in the search for novel and potent antibiotics. d) Using HPLC coupled with microbial assays, CA-ARS will compare antibiotic activities of individual food flavonoid compounds such as epigallocatgechin-3-gallate (green tea) and theaflavin-3-gallate (black tea) to activities of tea infusions with known composition of multiple compounds. This will determine whether anthocyanin and flavonoid levels of teas, wines, and fruit and vegetable juice predict biological potencies. e) Using the rainbow trout cancer bioassay, wherein fish are challenged with the carcinogen AFB1, CA-ARS and OR will work together to determine the cancer chemopreventive activities of the tomato glycoalkaloid tomatine. Various concentrations of tomatine will be added to diets along with AFB1, and reductions in tumor incidence in tomatine + AFB1 compared to AFB1 only will be used as the criterion for protection. Rodent studies will be conducted to confirm these observations. f) Crude extracts of a number of herbal and food products contain natural products that inhibit cancer cell growth and proliferation in a nuclear receptor dependent manner. Utilizing nuclear estrogen/androgen/Ah receptor cell and in vitro bioassay-directed fractionation approaches and instrumental analysis, CA-D will isolate and identify natural products responsible for this activity. The antiproliferative activity of the resulting extracts and chemicals will be examined using cancer cells in culture and promising candidate compounds will be analyzed in greater detail in the studies proposed in Objective 1. f) HI will will employ a bioassay that monitors cellular lipids, apolipoprotein expression and secretion in HepG2 cells to identify active anti-lipidemic and ant-diabetic components in the vegetable, Momordica charantia (commonly known as BM, bitter gourd, balsam pear or karela). Objective 4. Modifying foods is an increasingly important strategy to improve nutrition and safety. Therefore, we will improve food safety by developing approaches to increase beneficial or decrease adverse effects of bioactive food constituents and microbial contaminants. The following issues are proposed for research: a)GA-ARS will develop short-term mechanism-based in vivo rodent bioassays for assessing the efficacy of various processing methods to reduce the toxicity of DON and fumonisins in contaminated crops and foods used for human consumption. For DON, serum based metabolomic and proteomic markers will be identified in tissues and serum from untreated and DON treated B6C3F1 mice using data dependent liquid chromatography mass spectrometry/mass spectrometry. The DON work will be a collaborative effort with MI. For DON the endpoint will be changes in metabolite or protein profiles that correlate with the reduced weight gain that has been the hallmark of long-term DON exposure in mice and other animals. For fumonisins the endpoint will be alterations in metabolite profiles (altered sphingolipid metabolism) that correlate with renal toxicity in male Sprague Dawley rats. The bioassays will be used in combination with mass balance studies comparing toxin levels in wheat flour (containing DON) or corn flour (containing fumonisins), used to prepare selected food products using common cooking methods such as extrusion, baking, frying and nixtamalization. The levels of toxins in the processed foods will be compared with the toxicity of the processed foods in the short-term bioassay in order to ensure that the processing methods do in fact improve the safety of the final products. b) CA-ARS will develop improved processes for producing antimicrobial edible films from fruit and vegetable purees, and will determine whether low-temperature inactivation of pathogens in meat containing plant antimicrobials result in reduction in the levels of carcinogenic heterocyclic amines. c) UT will explore strategies to transform plants with enzymes to detoxify dietary carcinogens, like AFB1. In collaboration, OR has provided cDNA coding for rainbow trout CYP2K1, which converts AFB1 to an unstable epoxide that should readily react with abundant plant nucleophiles. Plants thus transformed would convert AFB1 contamination into safe endproducts. Production of plants such as maize transformed with genes coding for increased AFB1 detoxification pathways represents a potentially valuable pre-harvest AFB1 elimination strategy that would result in a safer food for animals and people. CO will provide additional expertise on transformation of plant models for this project. d) Using a poultry model for AFB1 hypersensitivity, UT and OR will test the effectiveness of chemopreventives shown in mammalian studies to protect against the toxic and carcinogenic activities of mycotoxins such as AFB1. These include, antioxidants shown to inhibit CYP-dependent AFB1 bioactivation, such as ethoxyquin, and agents shown to prevent absorption of AFB1 such as probiotic Lactobacillus rhamnosus. For those compounds that are shown to be protective, molecular mechanisms of protection will be explored. e) TX will continue to conduct animal studies and clinical intervention trials with NovaSil clay to confirm the safety of clay inclusion in the diet and the efficacy of aflatoxin enterosorption. Another aim of the laboratory is to understand the surface chemistry and molecular mechanisms involved in the interactions of other mycotoxins with diverse sorbents. Similar approaches are being used to develop and investigate novel porous solids for the sorption of arsenic and other priority contaminants of food and water. In ongoing collaborative research between OR and TX, OR will include NovaSil clay and combinations of clay and chlorophylls in cancer chemoprevention studies in trout. New collaborative studies will be intitated with USDA-GA and MI to apply the chemoprevention strategies for the mycotoxins fumonisin and deoxynivalenol using biomarkers identified in Obj. 2.

Measurement of Progress and Results

Outputs

• Research data, peer-reviewed publications, and presentations to the scientific and non-scientific community on:
• the anticarcinogenicity of chlorophylls, dietary indoles, dietary estrogens, and tea polyphenols and the assessment of potential adverse effects and development of potentially patentable uses for beneficial constituents.
• the anti-inflammatory, immune-enhancing and reproductive effects of phytochemicals, omega-3 fatty acids, dietary indoles, and dietary supplements and development of potentially patentable uses for beneficial constituents.
• the genotoxic, carcinogenic, immunotoxic and developmental effects of mycotoxins, phytoestrogens, plant alkaloids, and plant-derived cell-signaling modulators and development of patentable markers of exposure.
• bioassay-directed fractionation and identification of functional natural products and potentially patentable uses for newly discovered activities that protect both the food and consumers against human pathogens or other harmful constituents.
• Research data on the effects of pre- and post-harvest agricultural practices on beneficial or adverse effects of natural bioactive constituents, development of patentable processes and strategies for maximizing beneficial constituents and minimizing harmful constituents in foods, and development of surveys of exposure to beneficial and harmful constituents. Research tools have been made available for further study to other researchers; for example, purified natural chemicals, complex mixtures (extracts) with biological activity, biological reagents such as antibodies, cell lines, RNA from treated cells and laboratory animals.

Outcomes or Projected Impacts

• Improved consumer health, decreased human morbidity and mortality and reduced incidence of food poisoning as a result of increasing the beneficial properties of specific foods and food products and improved recommendations for specific dietary modifications by regulatory and authoritative agencies responsible for risk management decisions such as the USFDA, World Health Organization and the Food and Agriculture Organization.
• Increased consumer confidence through reduction in the uncertainty associated with risk analysis of bioactive food constituents and reduced economic costs for consumers and taxpayers due to increased certainty in the selection of agents for clinical intervention trials.
• Greater profitability for farmers, the American agrochemical industry, and food manufacturers by developing plant-food derived substances as safe, effective and economical antimicrobial and herbicidal agents and through the development of processing and food additives that reduce the presence or effects of harmful agents in commodities and food products.
• Greater profitability to the American pharmaceutical industry by developing novel specific bioactive plant constituents for use in cancer and disease therapy and prevention and the creation of mechanistic models for the development of safe, selective, and effective disease treatment and prevention.
• Improved research capability at university, government and industry research facilities resulting from the availability of improved purified natural chemicals, complex mixtures (extracts) with biological activity, biological reagents such as antibodies, cell lines and RNA from treated cells and laboratory animals.
• The productivity of the research groups is evident in the publication record documented at the W1122 WAAESD webpage (http://lgu.umd.edu/lgu_v2/homepages/pub.cfm?trackID=2154#5). Over the past 5 years the group has over 300 publications. A list of joint, collaborative publications between W-1122 colleagues is presented in the Attachment entitled "W-1122 Joint Publications.doc" A summary of relevant publications from each group is also provided in the attachments entitled "Recent publications W-1122.doc"

Milestones

(0): "W1122 Milestones.ppt" file in attachments

(0):lestones.ppt" file in attachments

Projected Participation

View Appendix E: Participation

Outreach Plan

New information and conceptual insights resulting from this research will be communicated in several ways. The full details of the research will be published in peer-reviewed scientific journals read worldwide. Other outreach avenues commonly used by W-1122 researchers include workshops and symposia at professional meetings ("Dietary Supplements: A Double-Edged Sword" at the 2005 Society of Toxicology Annual Conference), books on food safety ("Food Toxicology" published by CRC Press), and extension fact sheets in cooperation with Extension Specialists at Land Grant Universities. Another excellent example of W-1122 outreach is the Linus Pauling Institute (OR) which conducts a yearly international Conference entitled "Diet and Optimum Health," featuring lay-level addresses by noted researchers which are attended by hundreds (http://lpi.oregonstate.edu/meetings.html). A further venue for outreach is the Michigan State University National Food Safety and Toxicology Center (http://foodsafe.msu.edu/index.html) which conducts seminars and courses in food safety and risk assessment/management throughout the year. 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-1122 is in the process of establishing a website for rapid distribution of important information and for communication of constituents with W-1122 members.

Information resulting from investigations of W-1122 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-1122 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-1122 findings will be useful.

With the departure of extension specialist Dr. Carl Winter (CA) from this project, a gap has developed in our outreach capabilities. It will be a high priority to to recruit an extension food safety specialist to join this project. This task will be challenging, given the geographic diversity of W1122 members and the breadth of expertises that would need to be covered.

Organization/Governance

The Technical Committee consists of designated representatives from participating experiment stations, participants from Agricultural Research Service laboratories and the Administrative Advisor. An Executive Committee consists 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.

AUTHORIZATION: Electronic signature of the Administrative Advisor with submission data.

Literature Cited

Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S., and Vivanco, J.M. (2006) The role of root exudates in rhizosphere interactions with plants and other organisms. Annu. Rev. Plant Biol. 57:233-266

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 USA 98:14601-6.

Friedman, M., P. R. Henika, and R. E. Mandrell. (2002). Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica. J. Food Protection. 65:1545-1560.

Jeuken, A., Keser, B.J.G., Khan, E., Brouwer, A., Koeman, Jan and Denison, M.S. (2003) Activation of the Ah Receptor by Extracts of Dietary Herbal Supplements, Vegetables and Fruits, J. Agr. Food Chem. 51, 5478-5487.

Le, H.T., Schaldach, C.M., Firestone, G.L. and Bjeldanes, L.F. (2003) Plant derived 3,3-diindolylmethane is a strong androgen antagonist in human prostate cancer cells. J. Biol. Chem. 278:21136-21145.

Keck AS, Finley JW. (2004) Cruciferous vegetables: cancer protective mechanisms of glucosinolate hydrolysis products and selenium. Integr Cancer Ther. 3:5-12.

Lichtenstein P, Holm NV, Verkasalo PK, Iliadou A, Kaprio J, Koskenvuo M, Pukkala E, Skytthe A, Hemminki K. (2000) Environmental and heritable factors in the causation of cancer--analyses of cohorts of twins from Sweden, Denmark, and Finland. N Engl J Med. 343:78-85.

Riley, R.T. and Pestka, J. Mycotoxins: metabolism, mechanisms and biochemical markers. In The Mycotoxin Blue Book Duarte Diaz, Ed., pp 279-294. Nottingham University Press, Nottingham, UK.

Tilton SC, Givan SA, Pereira C, Bailey GS and Williams DE. (2006). Toxicogenomic profiling of the hepatic tumor promoters indole-3-carbinol, 17²-estradiol and ²-naphthoflavone in rainbow trout. Toxicol. Sci. 90: 61-72

Weisburger JH. (2000) Prevention of cancer and other chronic diseases worldwide based on sound mechanisms. Biofactors 12:73-81.

Yu, Z., Loehr, C., Fischer, K.A., Louderback, M., Krueger, S.K., Dashwood, R.H., Kerkvliet, N.I., Pereira, C.B., Jennings-Gee, J., Dance, S.T., Miller, M.S., Bailey, G.S. and Williams, D.E. (2006) In utero exposure of mice to dibenzo[a,l]pyrene produces lymphoma in the offspring: role of the aryl hydrocarbon receptor. Cancer Res. 66:755-762.

Attachments

Land Grant Participating States/Institutions

AZ, CA, CO, GU, HI, IL, MD, MI, NV, NY, OR, UT, VT

Non Land Grant Participating States/Institutions

NIFA, USDA-ARS/Georgia, USDA/ARS-California