W3122: Beneficial and Adverse Effects of Natural Chemicals on Human Health and Food Safety

(Multistate Research Project)

Status: Active

W3122: Beneficial and Adverse Effects of Natural Chemicals on Human Health and Food Safety

Duration: 10/01/2012 to 09/30/2017

Administrative Advisor(s):


NIFA Reps:


Statement of Issues and Justification

Overview. This application is a request to renew a productive regional project that has been in existence since 1971. Facilitated by comprehensive collaboration among participants, the overall goal of W-3122 is on improving food safety and human health worldwide. Research supported by W-3122 addresses the role that natural foodborne toxicants, cancer chemopreventives, botanical estrogens, dietary fiber, immune modulators and antimicrobials play in human health and disease. A particular strength of W-3122 is its dual focus on understanding the molecular basis by which dietary chemicals positively and negatively impact disease translating knowledge to improve human health by increased consumption of beneficial dietary chemicals and decreased intake of natural toxicants. These efforts cover a spectrum of cutting-edge scientific approaches that include gene expression, molecular signaling, genomics, tumor formation, and molecular biomarkers. The objectives of this renewal application continue W-3122's established emphasis on the role of natural dietary compounds in human health, while reflecting a continual evolution and refinement in the state-of-the-art. W-3122 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. Understanding the complex relationship between dietary chemicals and human health remains a paramount concern to public and agrifood industry stakeholders. These stakeholders include consumers, agricultural producers, food processors, health professionals, and policy makers charged with maintaining a safe and nutritious food supply. Dietary bioactive chemicals are defined in this proposal as naturally occurring substances produced by plants or microbes that exert beneficial or undesirable effects when they are consumed.

Excluding smoking, diet-related factors appear to 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).


There is also growing evidence that many dietary bioactive dietary chemicals can improve human health. For example, numerous epidemiologic studies indicate that regular consumption of several food commodities, especially fruits and vegetables rich in anticarcinogenic factors, and fiber-rich foods, 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. A specific example studied by W-3122 is chlorophyll and its derivatives, which can function as anticarcinogens and block the action of genotoxic chemicals such as aflatoxin B1. Bioactive dietary chemicals can affect other chronic diseases. With some exceptions, W-3122 research maintains that bioactive chemicals are most beneficial when present in the food matrix. This whole foods approach to disease prevention has been advocated by leaders of cancer chemoprevention, for example Thomas Kensler of Johns Hopkins University, who stated: Although pharmacological approaches establish proof of principle and help identify key molecular targets for interventions, food-based approaches that also use these molecular targets may be the most practical for widespread application in high risk populations (Kensler, et al., 2004). While W-3122 investigators have expended considerable research efforts in purified bioactive chemicals, these have been proof-of-concept studies that are often expanded into investigations of their effect in food matrices.

Our emerging focus on whole-foods has been affirmed by growing clinical evidence that vitamin supplements may do more harm than good (More Evidence Against Vitamin Use, New York Times, October 11, 2011). For example, in the Select Trial, vitamin E and selenium supplementation among 35,000 men led to a higher risk in prostate cancer (Klein, et al., 2011). Another study involving 38,000 women in Iowa discovered a higher risk of dying during a 19-year period among older women who used multivitamins and other supplements compared with women who did not (Neuhouser, et al., 2009). The findings translate to a 2.4 percent increase in absolute risk for multivitamin users, a 4 percent increase associated with vitamin B6, a 5.9 percent increase for folic acid, and increases of 3 to 4 percent in risk for those taking supplements of iron, folic acid, magnesium and zinc. Based on existing evidence, we see little justification for the general and widespread use of dietary supplements, the authors wrote. This increase in risk due to multivitamin consumptions is counter intuititve because this type of dietary intervention was previously viewed and an insurance policy to good health and these new findings suggest a significant risk to a practice that has previously been assumed as safe.

A January 2009 editorial in Journal of the National Cancer Institute noted that most studies of vitamins had shown no cancer benefits, but some had shown unexpected harms. Two studies of beta carotene found higher lung cancer rates, and another study suggested a higher risk of precancerous polyps among users of folic acid compared with those in a placebo group (Albanes, 2009).

In 2007, randomized trials of antioxidant supplements were extensively reviewed in a meta-analysis. In 47 trials involving 181,000 participants, the rate of dying was 5 percent higher among the antioxidant users. The main culprits were vitamin A, beta carotene and vitamin E whereas vitamin C and selenium seemed to have no meaningful effects Bjelakovic, et al., 2007).

Importance of Studying Dietary Bioactive Chemicals. Bioactive dietary chemicals are extraordinarily diverse with respect to chemical structures and biological activities. For example, aflatoxin B1 (AFB1) 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 another hazard due to its carcinogenic and teratogenic properties. Mycotoxins are not only carcinogens, but can suppress growth and immune function, rendering the consumer more susceptible to adventitious infections. Wide-spread Fusarium mold contamination of wheat and barley and resultant vomitoxin contamination often occur prompting food/cereal companies and the brewing industry to stop purchasing these commodities from U.S. farmers. 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-3122 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.

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 large human study in China showed that administration to humans of the chlorophyll derivative, chlorophyllin, could markedly reduce the level of DNA damage caused by AFB1. 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, or foods rich in these chemicals. Whether synthetic analogues of the Brassica indoles will have improved protective activities, is a subject of our further investigations.

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. Potential 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.

W-3122 researchers have determined that, in some cases, bioactive dietary chemicals have paradoxical effects. For example, the soy phytoestrogen and dietary supplement genistein, has been shown to reduce the number of DMBA-induced mammary tumors when administered to young rodents, probably through its estrogenic effect. However, when genistein is administered to rodents with estrogen responsive tumors, genistein enhances tumor growth. W-3122 researchers have also shown that Brassica indole, indole 3-carbinol (I3C), 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-3122 researchers.

Of particular relevance to this project, there is a burgeoning herbal product and food supplement industry in the U.S., recently estimated at $28 billion-a-year (Business Wire August 2011). The Dietary Supplement Health and Education Act by the U.S. Congress in October 1994 has blurred the distinction between 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, 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.

The primary foci of W-3122 are 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. Proposed work 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 Dietary Bioactive Chemicals. The research proposed herein is unique because exploits recent technical and conceptual advances in biomedicine within the context of agrigoods. 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-3122 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 (plants, human subjects, human and animal cell cultures, transgenic and knockout mice, rats, poultry, rainbow trout) with which to pursue this work. Using these models, members have established the benefits of chlorophylls, indoles, anti-oxidants, omega 3 fatty acids, fiber, various antimicrobials and plant growth factors. These models have also been used by W-3122 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.

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, 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-3122 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 members respective units. Their positions as faculty at major land-grant universities and USDA facilities ensure that data arising from W-3122 collaborative activities will be disseminated to the greatest extent possible among stakeholders and will thus provide maximum benefits to the U.S. public. W-3122'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 then help form the basis for an informed evolution of dietary habits to enhance or reduce the levels of these compounds thereby improving human health. Second, this research will result in the identification of new specialty crops and stimulation of the small farm economy. Third, 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 is generally safe, it is now known 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-3122 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.

Mycotoxins are common contaminants of many food crops including corn, wheat, barley and other important export commodities. Aflatoxins, found primarily in corn and peanuts, among other foods, are a major cause of liver cancer in many parts of the world, and is responsible for considerable losses to agriculture worldwide. Aflatoxin B1 (AFB1) is the most potent hepatocarcinogen known. W-3122 researchers have been at the forefront in determining mechanism of action, methods to reduce exposure, and using various chemopreventive strategies, such as chlorophylls, antioxidants, and probiotics, to reduce the human and animal diseases caused by this natural dietary carcinogen.

W-3122 researchers have developed a poultry model for extreme sensitivity to dietary carcinogens such as AFB1. This model has been rigorously validated, and gene products relevant to carcinogenesis have been characterized. These include cytochromes P450 1A4 and 3A4 that exhibit close similarities to human orthologs 1A2 and 3A4. These genes have been sequence and mapped, and expressed proteins have been made and validated for their similarity to human forms. Importantly, poultry models developed also show deficiencies and polymorphism in their glutathione S-transferase detoxification of AFB1, much in the same way as susceptible human populations. Because of these close similarities, it has been stated that the turkey may be a better model to study AFB1 carcinogenesis than rodent or other animal models (Rawal and Coulombe, 2011).

Fumonisins are primarily found in corn and corn-based foods and have been associated with several diseases in humans in areas of the world where corn is a major part of the diet. Fumonisin inhibition of ceramide synthase and the consequent disruption of lipid metabolism has been shown to be the underlying cause of all animal toxicity associated with fumonisins and/or infection with Fusarium verticillioides. Nonetheless, very little is known about the contribution of fumonisins to disease in humans even though they are suspected to be involved in liver and esophageal cancer (IARC, 2002), neural tube defects (Marasas et al., 2004) and stunting in infants (Kimanay et al., 2010). Recent work by members of W-3122 have identified high exposure populations in Guatemala and have initiated clinical and field studies using both exposure and mechanism-based biomarkers to reveal the involvement of fumonisins in human diseases and in particular neural tube defects. These studies show that elevated urinary fumonisin is significantly correlated with evidence of elevated sphingoid base 1-phosphates in blood spots. Future studies involving multiple members of W-3122 could provide a comprehensive approach to study the possible role of concurrent exposure to other mycotoxins found in corn including aflatoxin and deoxynivalenol all of which could conceivably contribute to the high incidence of stunting seen in infants in Guatemala.

The global incidence of Fusarium head blight (FHB) has increased markedly in recent years as a result of climate change, increased use of no-till farming to prevent soil erosion, use of susceptible high-yielding wheat and barley cultivars, non-optimal crop rotation and inadequate application of fungicide. Concurrent with increased FHB, there have been dramatic elevations in contamination by deoxynivalenol (DON or vomitoxin) and other 8-ketotrichothecene mycotoxins in cereal grains worldwide. The association of acute human illnesses in Asia with 8-ketotrichothecenes and the toxigenic fusaria that produce them has been well-documented. Cereal plants carry out phase II metabolism as a detoxification process with glucosylation being a primary reaction. DON can be glucosylated to DON-3-²-D-glucoside (D3G) in wheat, corn and barley suggesting that D3G and other 8-ketotrichothecene conjugates can contaminate human food Although trichothecene congeners are often detected in foods, toxicological research has focused primarily on a single toxin, DON, because it is found at the greatest concentrations. Research by W-3122 researchers has identified a unique receptor-mediated mechanism by which all trichothecenes are capable of inducing illness. Accordingly, there is need to both understand the mechanisms by which these mycotoxins cause acute and chronic health effect as well as the relative toxicologic potency of the different trichothecenes.This past year, GA-USDA, UT, and MI collaborated to publish a comprehensive review of the impact of exposure to mycotoxins (Riley, R.T., et al., 2011).

Natural products from plants and fungi have potent activities of many kinds and have been the primary sources of therapeutic agents for human maladies for millennia. The chemical diversity, structural complexity, and inherent biologic activity of natural products make them ideal candidates for new therapeutics. The primary focus of the research at CA-B laboratory has been to establish the efficacy and modes of action of certain indole phytochemicals from dietary plants as anticancer agents and therapeutic immune modulators. These studies have been recently broadened in an international collaborative project to screen Indonesian flora for natural products with these activities.

3,3-Diindolylmethane (DIM) is a promising antitumor agent derived from cancer protective Brassica vegetables, including cabbage, kale, broccoli, Brussels sprouts and several other commonly consumed vegetables. The anticarcinogenic effects of DIM have been established in animal models of spontaneous, carcinogen-induced or transplanted tumors. In addition, DIM, and its chemical precursor in the plants, I3C, has become the most popular adjunct therapies for recurrent respiratory papillomatosis, caused by certain types of human papillomaviruses. Results of our previous studies provide evidence that DIM modulates components of the immune response in cultured cells, which may be a key mode of action for the anti-cancer effects of this chemical. We showed that DIM could increase the expression of interferon gamma (IFN³) and IFN³ receptor in cultured cells, and potentiate the effects of IFN³ on the expression of other important components of the immune system. Oral DIM in mice increased the level of circulating IFN³ and other key immune signals, and strongly enhanced the proliferation of immune cells from the spleen. Moreover, DIM enhanced the clearance of an experimental virus from the gastrointestinal tract. These results strongly indicate that DIM functions as an immunomodulator, which may contribute to the anti-tumor activities of DIM.

Indoles (such as I3C) and isothiocyanates (sulforaphane, SFN) have been demonstrated to prevent cancer in a number of animal models and probably in humans. W-3122 researchers are examining how addition of these compounds, either as the pure supplement or as the whole food, to the maternal diet during pregnancy can protect the fetus from cancer later in life due to exposure in utero from chemical carcinogens capable of crossing the placenta. The model class of transplacental carcinogen that we have been studying are the polycyclic aromatic hydrocarbons (PAHs), formed from the burning of organic material (coal, diesel, tobacco, etc.). The majority of exposure (in non-smokers) to PAHs is through the diet. W-3122 research has shown that addition of I3C to the diet of a pregnant mouse provides significant protection for her offspring against PAH-induced cancers. Other studies currently underway will compare the pure I3C (available in stores or through the internet) to Brussels sprouts and to determine the mechanism(s) of this protection. The long-term goal is to translate this work to humans with the hope of providing phytochemicals or foods to a pregnant woman to reduce long-term chronic disease in her offspring.

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 (Helferich et al 2008). 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. Troublingly, there is a high incidence of self-medication with isoflavones in the US. Carefully controlled studies by W-3122 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. These important studies suggest that whereas soy consumption from an early age may be protective against breast cancer, short term, high-level consumption of the purified isoflavones in adulthood may promote cancer.

Autoimmune diseases, a constellation of chronic, disabling illnesses that result from the immune system at-tacking the bodys own tissues, are estimated to affect between 14 and 25 million persons in the U.S. Preclinical studies by W-3122 researchers have revealed that consumption of omega 3 fatty acids found in fish oil holds promise for preventing and ameliorating chronic autoimmune and inflammatory diseases including lupus nephritis. A CDC-NHIS survey determined that 11.7% of U.S. adults (26 million individuals) consume omega 3 supplements suggesting their already widespread usage (Barnes et al., 2004). The prevalence of autoimmune diseases is markedly impacted by environmental factors (e.g. toxicant exposures) and lifestyle choices (e.g. diet). For example, the risk of developing systemic lupus erythematosus (lupus), a prototypical autoimmune disease affecting 300,000 Americans and often associated with glomerulonephritis and kidney failure, is increased by occupational exposure to silica (e.g. farming, mining, construction, manufacturing and custodial industries. Consistent with these epidemiological findings, short-term inhalation exposure to silica accelerates the onset and severity of autoimmune nephritis in lupus-prone mice. W-3122 researchers are studying how this will specifically affect the triggering and exacerbation of autoimmune diseases by environmental toxicants.

Native Hawaiians and Pacific Islanders (NHPI) in Hawaii have more than twice the rate of obesity-associated type 2 diabetes (T2D) as compared to Caucasians and more than five times as likely to die from T2D. There is a growing awareness and mounting body of scientific evidence that successful implementation of strategies to control T2D among ethnic minorities will require culturally appropriate interventions. Morinda Citrifolia L. (Rubiaceace) commonly known as noni and Momordica charantia (bitter melon, BM) is native to Polynesia and widely cultivated in tropical areas including Hawaii. Mechanistic studies indicate a role for chronic inflammation in pathophysiology of obesity and T2D. Furthermore, an imbalance in gut microbiota, resulting in inflammation has been implicated in T2D and obesity. W-3122 researchers have recently demonstrated that both, noni and BM improve glucose metabolism and T2D in mice fed a high-fat diet (HFD). Current studies are directed to investigate the anti-inflammatory properties of noni and BM as well as effects of BM on gut microbiota in mice fed a HFD.

Dysbiosis of gut microflora has been linked to a growing number of inflammatory and chronic disease states, including colon cancer (Sobhani et al. 2011), IBS (Codling et al. 2010), and rheumatoid arthritis (Scher and Abramson, 2011). Thus, potential modulators of our commensal microflora, such as probiotics and prebiotics, are receiving considerable attention. Probiotics are live ingested organisms that assist with these beneficial functions, while prebiotics are dietary components that encourage the growth of beneficial organisms in the gut. Fermented foods often serve as a delivery vehicle for both pre- and probiotic components and new research is focusing on how the fermentation process alters bioactive dietary components both ex vivo (fermented foods) and in vivo (gut fermentation) and affects native microflora populations. W-3122 investigators are demonstrating that dietary intake of rice bran alters fecal levels of Lactobacillli, as well as several bacteria that produce butyrate, which feeds and protects the colon. Rice bran consumption also decreases Bacteriodes spp. associated with colon cancer and gut inflammation. Rice bran is a rich source of bioactive tocopherols, polyphenols, and sitosterols we are also exploring the role of both probiotic organisms (Ryan et al., 2011) and commensals in altering the levels and bioavailability of these compounds.

Nuclear hormone receptors are ligand-dependent transcription factors that play key regulatory roles in controlling both normal physiological processes mediated by endogenous chemicals (hormones), but these same receptors can exert significant modulatory effects on human disease processes and progression. Dietary chemicals have been shown to produce estrogenic and antiestrogenic effects and stimulate/inhibit cell (tumor) growth though their ability to bind to and activate/inhibit estrogen receptors (Safe and Papineni, 2006). Accordingly, W3122 investigators have developed nuclear receptor based cell bioassay systems for high-throughput screening of chemicals and food and environmental extracts for the nuclear receptor agonist/antagonist activities. Natural and dietary chemicals have been shown to bind to and activate/inhibit estrogen receptors resulting estrogenic and antiestrogenic effects in vitro and in vivo and the production of adverse or beneficial effects (Safe and Papineni, 2006). The documented ability of these and related nuclear receptors (i.e. the Ah receptor) to bind and be activated/inhibited by structurally diverse chemicals from food and environmental samples suggests that there are a large number of natural and synthetic bioactive compounds that can bind to and/or activate/inhibit these receptors and receptor signal transduction pathways (Hilscherova et al., 2000; Denison and Nagy, 2003). The application of receptor bioassay-based fractionation approaches coupled with instrumental analysis methods have been extremely successful in isolating and identifying bioactive chemicals present in food extracts containing complex mixtures of chemicals (Hilscherova et al., 2000). These bioassay-based screening approaches will continue to allow isolation and identification of bioactive chemicals present in food and other matrices that can could produce adverse endocrine disrupting effects in exposed individuals, but also that can be used to develop therapeutic agents for human disease (i.e. breast and other hormone-dependent cancers).

W-3122 researchers have pioneered methods to inactivate bacterial toxins through confirmation changes in foods by plant compounds. Changes of the native structural integrity may inactivate the toxin by preventing molecular interaction with cell membrane receptor sites of their host cells. In ongoing collaborative studies, W-3122 researchers found that apple juice inhibited the biological activity of virulent Shiga toxin Stx2. Additional studies with immunomagnetic beads bearing specific antibodies against the toxin revealed that the Stx2 added to apple juice appears to be bound to the apple juice constituents, which are believed to be phenolic and anthocyanin compounds. We also found that apple juice also inactivated enterotoxin A produced by Staphylococcus aureus (Quinones et al., 2009). These results suggest that food-compatible and safe anti-toxin compounds can be used to inactivate Shiga and other toxins in vitro and possibly also in vivo. Possible mechanisms of toxin inactivation are not known.

Objectives

  1. Determine the mechanisms by which dietary bioactive compounds protect against human diseases.
  2. Elucidate mechanisms of action of dietary toxicants and develop biomarkers for human risk assessment and disease prevention.
  3. Discover and characterize novel bioactive dietary compounds that have beneficial or adverse effects on human health.
  4. Increase beneficial or decrease adverse effects of bioactive constituents and microbes in food.

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-3122 scientists. Several of the major active and planned collaborative studies among W-3122 scientists have been summarized (Table 1), along with the special contributing research capabilities of the different sites (Table 2). All data will be shared among all project members and summarized activities will be accessible to the public via a W-3122 web site. Objective 1. Determine mechanisms of action by which dietary bioactive compounds protect against human diseases. The following areas are proposed for further research: a) CO will use microbiome sequencing and qPCR to determine how dietary components affect the dynamics of hostassociated microbial communities. They will also use global metabolomic profiling, utilizing both GC- and UPLC-MS, to identify the changes in chemical composition of fermented foods and to quantify levels of bioactive plant and microbial metabolites in plasma, tissue, fecal, and urine samples. In vitro bioassays and mouse models will be used to determine the anticancer and antimicrobial effects of extracts from fermented and nonfermented foods and PCR-based microarrays, gene expression analysis, and immunotyping by flow cytometry will be used to explore potential molecular targets and mechanisms. Current research focus areas are rice bran, tea, and dry bean. In addition, CO will utilize metabolomic and microbiome analysis in collaboration with HI to identify the organisms involved in the fermentation of noni juice and how that affects levels of bioactive noni polyphenols. b) UT will assess the potential chemoprotective properties of probiotics against AFB1 toxicity in poultry in collaboration with OR and CO. In two clinical studies, probiotics have reduced AFB biomarkers in humans. In addition to pathology endpoints (markers of liver function), we will perform microarray and conduct a complete transcriptome analysis of gene transcripts important in cancer development and chemoprevention. UT will also assess impairment of cellular and humoral immunity in this model in collaboration with MI. c) CA-B will use gene expression microarray and immuno-protein array techniques to determine the kinetics and concentration-dependent effects of the dietary indole, DIM, and related compounds, on the expression in cultured tumor cells and immune cells of genes and proteins associated with immune regulation of tumor growth. 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 immune response and tumor development will be assessed in rodents. CA-B will continue to cooperate with CA-D and MI in studies of the role of the Ah receptor in the immune enhancing effects of the Brassica indoles. d) CA-D will use a combination of in vitro and recombinant cell-based bioassays to examine the mechanisms by which dietary bioactive chemicals/extracts can inhibit proliferation of cancer cells. The ability of these chemicals/extracts to inhibit the proliferation of breast and liver cancer cells will be determined using the sulforhodamine B assay and positives will be further examined for their ability to activate/inhibit the functionality activity of Ah and estrogen receptors (key players in cell proliferation responses). Direct interactions of the chemicals/extracts with these receptors will also be examined using ligand- and DNA-binding and receptor-dependent reporter gene assays. Demonstration of the ability of these chemicals/extracts to inhibit these receptor pathways in vivo will be carried out in mice in collaboration with UI and effects on other nuclear receptor pathways examined using bioassays developed at UN-R. e) IL will utilize in vitro approaches to evaluate mechanisms by which botanical estrogens act on estrogen responsive breast cancer cells. This team will examine the ability of these biologically active compounds to bind to the estrogen receptors (ER). Additionally, they will determine binding affinities to both ER-alpha and ER-beta and how binding to these receptors can alter gene expression - especially those related to estrogen function, cell proliferation, and apoptosis. f) MI will study the effectiveness on omega-3 fatty acids for mitigating toxicant- accelerated lupus nephritis as well as ascertain the appropriate dietary omega-3s levels required to counter triggering and exacerbation of lupus nephritis and other autoimmune diseases. g) OR will study a pregnant mouse model for transplacental cancer chemoprevention studies. The phytochemical is added to the diet of the pregnant mouse and fed throughout gestation and lactation. During gestation, the mouse is exposed to PAHs which cross the placenta and induce a variety of cancers in the offspring (lymphomas, lung, liver, ovarian) when they reach adulthood. OSU will study alterations in gene expression in the fetus when the mother is fed I3C. This information may help us determine if the mechanism(s) of this protection will be applicable to humans. There will be particular focus on alterations in the epigenome as the fetus is known to be very susceptible to diet-induced epigenetic alterations. h) HI will elucidate anti-inflammatory mechanisms will be elucidated in liver, adipose and intestine of mice fed HFD with either noni or BM using real-time quantitative PCR, Western blotting, enzyme assays, siRNA techniques as well as gel-shift assays and chromatin immunoprecipitation (ChIP) assays. Objective 2 Elucidate mechanisms of action of dietary toxicants and develop biomarkers for human risk assessment and disease prevention. The following issues are proposed for further research: a) UT will study the effect of AFB1 on various phase I and II genes relevant to susceptibility and resistance to liver cancer in poultry exposed to dietary AFB1. Biomarkers for molecular effect will be evaluated and validated. Effects on genes relevant to susceptibility to carcinogenesis include those related to phase I and phase II metabolism, inflammation, cell response, apoptosis, and expression of activated p53 protein will be measured. In collaboration with OR and CA-D, UT will investigate the regulation of GSTs, universal cancer protective enzymes in the poultry model to gain insight on the role of this enzyme in dietary induced carcinogenesis in people. b) MI will used cloned cell lines and animal models for anorexia and emesis to understand the role of bitter taste receptors in mediating gastroenteritis and growth suppression by trichothecene mycotoxin congeners and other dietary toxicants. MI will cooperate with GA-USDA to determine how processing affects toxicity of the trichothecenes and reflected by cellular and animal models. c) GA-USDA will conduct dose-response studies to determine the minimum oral dose of FB1 that disrupts sphingolipid metabolism and induces toxicity (increased apoptosis) in rat kidney and mouse liver. We will also determine the minimum dose of FB that will cause a significant elevation of FB in urine and sphinganine 1-P and 1-deoxysphinganine in blood spots and we will compare the sphingolipid metabolite profiles in target tissues in rat and mouse. d) GAUSDA will determine the dietary no observed effect and lowest observed effect levels for neural tube defect induction and determine the dose-response thresholds for elevation in sphingolipid biomarkers in blood spots and fumonisins in urine using animal models. This will be accomplished using three mouse studies with oral dosing of FB1. The first two will be gavage studies and the third will be a feeding study using diets containing FB. e) GAUSDA will determine the relationship between fumonisin consumption, urinary fumonisin (exposure biomarker) and changes in sphingolipids in blood spots (effect biomarker) in human populations consuming corn. This will be accomplished by identifing individuals consuming large amounts of corn-based foods in communities where FB is infrequently detected (low exposure) and frequently detected (high exposure) and sample and analyze urine (FBs) and blood spots (sphingolipids). f) CA-D will use a series of recombinant cell-based bioassays to determine the ability of various plant extracts to activate or inhibit gene expression mediated by nuclear receptors (i.e. Ah and steroid hormone receptors). Positive extracts will be fractionated using conventional chromatographic methods, the desired bioactivity of fractions determined using the cell bioassays and active fractions subjected to further separation and tested in the bioassays. This bioassay-directed fractionation approach will lead to isolation and purification of the responsible bioactive chemical(s) and its identity determined by instrumental analysis methods. The responsible chemicals can be further characterized in vivo in receptor-responsive bioassays in mice and collaboration IL or OR or further characterized with NV using a battery of other in vitro nuclear receptor bioassays. Objective 3. Discover and characterize novel bioactive compounds that have beneficial or adverse effects on human health. The following issues are proposed for further research: a) IL will use a variety of in vivo and in vitro techniques to determine the role of botanical estrogens on growth of cultured estrogen-responsive breast tumor (MCF-7) cells and changes in gene expression patterns associated with exposure to the botanical estrogens. The IL will also conduct in vivo investigations to evaluate the effect of the dietary botanical estrogens on estrogen-dependent tumor growth. Additional in vivo investigations will focus on prevention of breast progression. In these investigations, metastatic BC engineered to express luciferases will be used to follow growth and metastatic progress in real time. When mice are injected with luciferin, the tumor cells will emit light that can be detected by bioluminescent imaging (BLI). b) CO and HI will collaborate on determining the effects of noni juice chemical constituents and explore how bitter melon consumption alters obesity-associated gut microflora in mice fed a high fat diet. c) NV will use cell culture and animal models to further elucidate the underlying molecular mechanism behind the triglyceride lowering ability of a grape seed procyanidin extract (GSPE). Cell culture models will be used for transient transfection studies to determine whether or not GSPE enhances the recruitment of co-activators to the FXR-CDCA-GSPE complex and if this explains the enhanced transactivation of FXR seen in the presence of GSPE. NV will use rodent models, including mice and rats will be utilized to determine the ability of GSPE to lower serum triglyceride levels in a hypertriglyceridemic state, e.g. following fructose consumption, as well as in conditions under which circulating bile acid levels have been reduced, namely by treatment with a cholestyramine diet or via surgical ligation of the common bile duct. Hepatic gene expression analysis will also be conducted under these experimental conditions to determine changes exerted by GSPE at the molecular level. Gene expression microarray analysis will also be conducted to determine any gene expression changes in the intestine, a well-known FXR target tissue. d) To further understand the beneficial actions of the whole grape seed procyanidin extract, NV will fractionate components in the extract and using transient transfection assays we would initially assess the ability of the isolated fractions to enhance the transactivation of FXR, as is observed with the whole extract. The most potent components within the extract responsible for the triglyceride lowering effect of GSPE would be identified. Subsequently follow-up studies would include testing the actions of those bioactive fractions in vivo in wild type and FXR knockout mouse models. e) CA-B, MI and CA-D will collaborate on determining immune modulating effects of dietary indoles and natural products from Indonesian plants and microbes. The initial screens by CA-B of Indonesian flora will employ high-throughput, cell-based assays of extracts for anticancer and immune modulating activities. Following the activity-based chromatographic purification and spectral identification of active substances, the activities of purified compounds will be characterized more fully in studies to include rodent assays. For studies of the modes of action of the indoles and other purified natural products, CA-B will use gene expression microarray and immuno-protein array techniques to determine the kinetics and concentration-dependent effects of the purified compounds, on the expression in cultured tumor cells and immune cells of genes and proteins associated with tumor growth and immune regulation. Promoter analyses will be conducted of key genes implicated in anti-tumor progression activities of compounds to identify regulatory mechanisms for this process. The functional effects of implicated genes on in vitro and in vivo measures of immune response and tumor development will be assessed in rodents. CA-B will continue to cooperate with CA-D and MI in studies of the role of the Ah receptor in the immune enhancing effects of the Brassica indoles and other natural products. f) CA-USDA will conduct collaborative studies using bacterial assays and mass spectrometry, to screen plant extracts, spice powders and essential oils capacity to chemically inactivate E. coli O157:H7 and decrease formation of heterocyclic amines in beef patties (hamburgers) and mutagenic and carcinogenic HCAs (MeIQx and PhIP) formed during grilling of ground beef. CA-USDA will also investigate the kinetics of the compounds tested to determine their interaction in the meat matrix when exposed to heat; and (b) to confirm the beneficial effects and safety of the added compounds by animal feeding studies. These natural and safe plant-derived formulations will be used for their beneficial antimicrobial and anti-heterocyclic amine effects in grilled meats. g) MI will conduct modeling studies to determine the relative potencies of the different trichothecenes in neurotoxic and immunotoxic endpoints to generate toxic potency factors that can be applied to risk assessment. MI will cooperate with HI to determine the role of bitter taste receptors in mediating anti-obesity and anti-diabetes effects of natural compounds in foods and supplements. MI will cooperate with CA and CA-B in determining bitter taste agonism in natural toxic and bioactive compounds. h) HI will conduct metabolomics and micobiome analysis techniques in collaboration with CO to identify active components of noni and the effect of BM on gut flora. OBJECTIVE 4. Increase beneficial or decrease adverse effects of dietary constituents and microbes. The following issues are proposed for further research: a) GA-USDA will determine the specific mechanism(s) by which fumonisins are readily taken up by corn plant roots and yet have limited translocation into above ground vegetative tissues. Lack of knowledge about how the plant/fungal interaction influences FB production and F. verticilliodes plant diseases is a major impediment to developing new strategies to reduce FB contamination in corn. Studies will be conducted to determine if A) FB1 affects plant transpiration in a dose-dependent manner, B) sphingoid bases and their 1-phosphates are mobile metabolites moving from roots to aerial tissues via transpiration processes, and (C) these parameters will differ between FB1-sensitive and -insensitive genotypes of corn. b) GA-USDA will determine the ability of alkaline cooking of maize to reduce free FB concentration and FB-related toxicity of whole kernel corn using a proven rat feeding bioassay. Researchers will utilize naturally contaminated corn (<10 ¼g FB1/g) and food grade lime so that experimental conditions are relevant to households and the food industry. FB-contaminated (> 4, < 10 ¼g FB1/kg) whole kernel corn will be cooked (90 - 99 C) and steeped in the alkaline water prepared from tap water and food grade lime purchased locally (pH >10.5). After steeping, the corn will be rinsed with water, freeze dried and ground. Various recipes (water: corn ratio, lime concentration, cooking time, number of rinses, etc) will be compared to determine the conditions maximizing FB1 reduction. c) CA-USDA will determine whether plant compounds can concurrently inactivate both E. coli O157:H7 using bacterial assay and Shiga toxins using the MTT assay. The MTT assay for inhibition of biological activity of the toxin is based on its ability to inhibit protein synthesis and mitochondrial dehydrogenase activity in the Vero cells. Toxin activity is therefore inversely related to the population of the cells, i.e., inactivation of Stx2 is measured by increased % cell survival. The proposed strategies to overcome the virulence of bacterial toxins are expected to result in the discovery of food-compatible formulations active against both foodborne pathogens and toxins produced by these pathogens that will benefit microbial food safety, animal and human health, food security, and the economy.

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.
  • Inform the American food industry on potential bioactive chemicals and fiber present in food ingredients that can benefit consumer health.
  • Identification of novel value added crops and foods that can be exploited by farmers and processors, respectively
  • 6. Dissemination of data in scientific meetings and peer-reviewed journals 7. 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-3122 researchers.

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 autoimmune diseases and immune suppression.
  • 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.
  • Informing 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): attached ppt: Milestone

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-3122 is in the process of establishing a website for rapid distribution of important information and for communication of constituents with W-3122 members.

Information resulting from investigations of W-3122 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-3122 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-3122 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

IARC (2002) IARC Working Group on the Evaluation of Carcinogenic Risk to Humans. Vol. 82, Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene, Lyon, IARC Press, pp301-366.

Kimanya, M. E., De Meulenaer, B., Roberfroid, D., Lachat, C., & Kolsteren, P. (2010). Fumonisin exposure through maize in complementary foods is inversely associated with linear growth of infants in Tanzania. Mol Nutr Food Res., 54 (11), 1659-67.

Marasas, W.F.O., Riley, R.T., Hendricks, K.A., Stevens, V.L., Sadler, T.W., Gelineau-van Waes, J., Missmer, S.A., Cabrera Valverde, J., Torres, O.L., Gelderblom, W., Allegood, J., Martínez de Figueroa, A.C., Maddox, J., Miller, J.D., Starr, L., Sullards, M.C., Roman Trigo, A.V., Voss, K.A., Wang, E., Merrill, A.H., Jr. (2004) Fumonisins disrupt sphingolipid metabolism, folate transport and development of neural crest cells in embryo culture and in vivo: A risk factor for human neural tube defects among populations consuming fumonisin-contaminated maize? Journal of Nutrition. 134:711-716.

Quiñones B, Massey S, Friedman M, Swimley MS, Teter K. 2009. Novel cell-based method to detect Shiga toxin 2 from Escherichia coli O157:H7 and inhibitors of toxin activity. Applied and Environmental Microbiology 75(5):1410-1416.

Sobhani, I., et al., Microbial Dysbiosis in Colorectal Cancer (CRC) Patients. PLoS ONE, 2011. 6(1): p. e16393.

Codling, C., et al., A Molecular Analysis of Fecal and Mucosal Bacterial Communities in Irritable Bowel Syndrome. Digestive Diseases and Sciences, 2010. 55(2): p. 392-397.

Scher, J.U. and S.B. Abramson, The microbiome and rheumatoid arthritis. Nat Rev Rheumatol, 2011. 7(10): p. 569-578.

Ryan, E.P., et al., Rice Bran Fermented with Saccharomyces boulardii Generates Novel Metabolite Profiles with Bioactivity. Journal of Agricultural and Food Chemistry, 2011. 59(5): p. 1862-1870.

Helferich, WG, et al., (2008) Phytoestrogens and breast cancer: a complex story. Immunopharmacology. 16: 1-8.

Del Bas JM et al., Dietary procyanidins lower triglyceride levels signaling through the nuclear receptor small heterodimer partner Mol Nutr Food Res, 2008, 52: 1172-1181.

Del Bas JM et al., Dietary procyanidins enhance transcriptional activity of bile-acid activated FXR in vitro and reduce triglyceridemia in vivo in an FXR-dependent manner Mol Nutr Food Res, 2009, 53:805-814.

Watanabe M, et al., Bile acids lower triglyceride levels via a pathway involving FXR, SHP and SREBP1c. JCI, 2004, 113: 1408-1418.

Safe, S. and Papineni, S. (2006) The role of xenoestrogenic compounds in the development of breast cancer. Trends in Pharmacol. Sci. 27, 447-454.

Hilscherova, K., Machala, M., Kannan, K., Blankenship, A.L. and Giesy, J.P. (2000) Cell bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediated activity in environmental samples. Environ. Sci. Pollut. Res. Int. 7, 159-171.
Denison, M.S. and Nagy, S.R. (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals, Ann. Rev. Pharmacol. Toxicol. 43, 309-334.

Riley, R.T., Voss, K.A., Coulombe, R.A., Pestka, J.J. and Williams, D.E. 2011 Developing Mechanism-Based and Exposure Biomarkers for Mycotoxins in Animals, in Determining Myctotoxins and Mycotoxigenic Fungi in Food and Feed, S. DeSaeger (ed.), Woodhead, Publ., Cambridge, U.K.)

Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. JAMA. 2007 Feb 28;297(8):842-57.

Kensler TW, Egner PA, Wang JB, Zhu YR, Zhang BC, Lu PX, Chen JG, Qian GS, Kuang SY, Jackson PE, Gange SJ, Jacobson LP, Muñoz A, Groopman JD. Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas. Gastroenterology. 2004 Nov;127(5 Suppl 1):S310-8.
Klein EA, Thompson IM Jr, Tangen CM, Crowley JJ, Lucia MS, Goodman PJ, Minasian LM, Ford LG, Parnes HL, Gaziano JM, Karp DD, Lieber MM, Walther PJ, Klotz L, Parsons JK, Chin JL, Darke AK, Lippman SM, Goodman GE, Meyskens FL Jr, Baker LH. Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA. 2011 Oct 12;306(14):1549-56.


Neuhouser ML, Wassertheil-Smoller S, Thomson C, Aragaki A, Anderson GL, Manson JE, Patterson RE, Rohan TE, van Horn L, Shikany JM, Thomas A, LaCroix A, Prentice RL. Multivitamin use and risk of cancer and cardiovascular disease in the Women's Health Initiative cohorts. Arch Intern Med. 2009 Feb 9;169(3):294-304.

Rawal S, Coulombe RA Jr. Metabolism of aflatoxin B1 in turkey liver microsomes: the relative roles of cytochromes P450 1A5 and 3A37. Toxicol Appl Pharmacol. 2011 Aug 1;254(3):349-54.

Attachments

Land Grant Participating States/Institutions

AZ, CA, CO, HI, IL, MI, MS, ND, NJ, NM, NV, OR, TX, UT, VT, WA, WI

Non Land Grant Participating States/Institutions

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