W4122: Beneficial and Adverse Effects of Natural Chemicals on Human Health and Food Safety
(Multistate Research Project)
W4122: Beneficial and Adverse Effects of Natural Chemicals on Human Health and Food Safety
Duration: 10/01/2017 to 09/30/2022
Statement of Issues and Justification
Overview. This application is a renewal of a productive regional project that was started in 1971. The overall goal of W-3122 researchers is to examine the effects that bioactive components of the diet such as phytochemicals, foodborne toxicants, microbial metabolites, and specific macro- and micronutrients exert on human health and in the safety of the food supply. W-3122 participants collectively utilize mechanistic, preclinical, and clinical research methods to provide a comprehensive translational approach towards understanding the role of natural chemicals in human health and food safety. These efforts include use of cutting-edge research methodologies to address a broad range of research questions. Topics addressed include examination of the effects of whole foods and specific dietary components on gut ecology, understanding the molecular basis of both carcinogenesis from food-borne toxicants and chemo-protection by beneficial dietary chemicals, effects of food processing on bioactivity and bioavailability of food-borne chemicals, and trans-generational health effects of dietary and environmental exposures. The objectives of this renewal application represent our continued commitment to understanding the relationship between dietary components and human health while emphasizing emerging areas of scientific inquiry, such as the interplay between dietary chemicals and the gut microbiome and dietary regulation of the host epigenome. W3122 was selected for the Western Region Award of Excellence in 2015 and 2016, and has been highly successful as measured by numerous collaborative efforts, extensive publications and other outreach initiatives such as presentation of lectures and development of websites and curriculum modules. We anticipate that this renewal project will be equally successful and will continue to have an impact on issues related to food safety and human health.
Stakeholders. 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 or produced by human or microbial metabolism in the body. How these chemicals influence human health, disease development, and food safety is important to everyone. Understanding how to enhance the benefits or minimize the risks of specific dietary compounds is important for agricultural producers, food processors, healthcare professionals, and policy makers charged with determining optimal human nutrition requirements and maintaining the safety of the food supply.
Rationale. Natural chemicals consumed in the diet have the ability to positively or negatively impact human health. Phytochemicals found in fruits in vegetables can reduce disease risk by acting as anti-oxidants, hormone mimics, signaling molecules, and modifiers of the host epigenome and gut microbiota. Conversely, consumption of certain types of sugars and fats can increase inflammation, lead to reduced insulin sensitivity, and increase the risk of developing obesity and chronic disease. Microbial metabolites found in food or produced from its fermentation can also exert effects on human health. Beneficial microbial metabolites include short chain fatty acids, which act as cellular signals to modulate host metabolism and serve as energy for colonic epithelial cells, and products of phytochemical catabolism which may have increased bioactivity and bioavailability relative to their parent compounds. Microbial metabolites also include fungal and bacterial toxins, such as fumosin and aflatoxin, which are harmful to human health. Determining dietary exposure to these toxins and identifying their molecular targets in human hosts is critical in establishing acceptable exposure levels and ensuring a safe food supply. In general, in order to understand the particular benefits or risks of a given dietary chemical, it is necessary to understand dietary exposure levels, bioactive doses, factors influencing absorption and metabolism, molecular targets in the body, synergistic effects with other compounds and trans-generational effects. W-122 researchers are actively engaged in exploring these facets of dietary chemicals, generally in the context of their role in whole foods.
The rationale for a whole foods approach is multifaceted. Dietary components may be more easily absorbed by the body when present in their natural matrix and can interact with other compounds in whole foods to have increased benefits. Consuming bioactive dietary compounds as a whole food can also minimize the risk of reaching toxic or detrimental dose levels and whole foods are also more cost effective than concentrated dietary supplements. Finally, evidence from a number of clinical trials and epidemiological studies suggest that consuming whole foods is more efficacious in disease prevention than consuming supplements containing specific bioactive compounds. A recent meta-analysis of clinical trials exploring cardiovascular benefits of consuming lycopene in tomato-based foods versus synthetic supplements supports consuming tomato-based foods as a first line approach to preventing cardiovascular disease (Burton-Freeman and Sesso, 2014). W-122 researcher, Helferich and colleagues have shown in mouse models of breast cancer that soy flour has opposite effects of consuming isoflavone supplements, suggesting that the soy flour may be beneficial while supplementation could increase disease risk (Liu et al., 2015). Another recent study examined effects of various fiber types on enhancing the growth of specific members of the gut microbiota and enhancing the protective mucosal lining of colonic epithelial cells. These researchers noted that consumption of complex plant polysaccharides showed more benefits for intestinal health and pathogen protection than consumption of diets supplemented with specific purified prebiotic fibers (Desai et al., 2016).
Dietary interactions with the gut microbiome. W-122 researchers have an established record of exploring mechanisms of action of beneficial and harmful dietary chemicals and for exploring ways to mitigate or enhance their effects through agricultural practices or food processing. However, the advent of new sequencing technologies has allowed us to identify and examine how the trillions of microorganisms in our intestines contribute to host health and physiology. It has been established that these organisms are critical to digestion, pathogen protection, and immune modulation (Sekirov et al. 2010). An imbalance, or dysbiosis, of the microbiota has been associated with inflammatory diseases of the intestines but also with cardiometabolic dysfunction like Type 2 diabetes and heart disease (Festi et al., 2014) and with autoimmune conditions like rheumatoid arthritis (Wu et al., 2016) and Parkinson’s disease (Sampson et al., 2016). Several mechanisms have linking microbiota, diet, and disease development or prevention are being established. One prevalent and well-supported hypothesis suggests that high fat diet induced microbial dysbiosis is associated with loss of integrity of the intestinal epithelial barrier and translocation of bacterial components such as lipopolysaccharides (ie. bacterial endotoxin), which results in a condition referred to as metabolic endotoxemia (Cani et al. 2007). Metabolic endotoxemia is associated with chronic low-grade inflammatory processes that contribute to various components of cardio-metabolic disease.
Specific microbial metabolites of dietary components are also key modulators of host disease processes. Dietary fiber serves as food for the colonic bacteria and is fermented to short chain fatty acids such as butyrate, proprionate, and acetate. These products can interact with free fatty acid receptors in the gut, liver, and adipose tissue to regulate intestinal transit time and glucose and lipid storage (Kasubuchi et al. 2015). Butyrate serves as the primary food source for colonic epithelial cells and is though to have anti-tumorogenic effects by acting as an HDAC inhibitor (Davie, 2002). It has also been shown that butyrate is critical in maintaining hypoxic conditions at the epithelium-lumen interface and stabilizing the expression of Hypoxic Inducible Factor (HIF-1a), which regulates tight junctions between epithelial cells (Kelly et al. 2015). Conversely, other metabolites produced by microbial processes can have detrimental effects to the host. Protein degradation by colonic bacteria is associated with production of pro-carcinogenic metabolites such as N-nitroso compounds and hydrogen sulfides (Hughes et al. 2000). Choline and carnitine consumption are associated with microbial production of trimethylamine oxide (TMAO) which can interfere with reverse cholesterol transport processes and result in development of atherosclerotic plaques (Koeth et al. 2013). Therefore, understanding the influence of diet on the microbiota and microbial processes is emerging as an important aspect of understanding how dietary chemicals can influence or prevent certain diseases. W-122 researchers are making important contributions to this area, particularly with respect to understanding how dietary microbiota manipulation can be used to prevent colorectal cancer and metabolic syndrome.
Bioactive dietary chemicals as epigenetic regulators. Another area of research that is gaining attention is the regulation of gene expression by dietary components via epigenetic mechanisms. As we learn more about human genetics, it is becoming increasingly clear that certain gene variants are associated with higher risk of developing obesity and chronic diseases such as cancer and cardiometabolic diseases. However, while genetic factors can increase disease risk, environmental factors can modulate the expression of these genes through epigenetic mechanisms. Dietary components choline and betaine can act as methyl donors to epigenetically tag genes for silencing. However, other bioactive dietary compounds may act in more subtle ways (such as regulating acetylation of histones) to influence epigenetically regulated gene expression. Understanding how these dietary components interact with the epigenome will be key in understanding how various bioactive dietary components can influence disease development or prevention. W-122 researchers are examining questions such as how maternal exposure to common environmental toxins influence disease development in offspring and how dietary bioactive compounds, such as certain phytochemicals might act via the epigenome to mitigate these risks. Epigenetic imprinting is most marked in the prenatal and early post-natal period and identifying ways to mitigate disease in later life may rely, in part, on proper nutrition during these periods. However, the epigenome can be modified throughout the lifespan of an individual and W-122 researchers are also exploring how dietary modifications influence the epigenome in adult organisms for the mitigation of risk of developing colorectal cancer and other diseases. Epigenetic regulation has not been extensively examined in the context of dietary interventions and may represent a novel means of mitigating disease risk, particularly in genetically predisposed individuals.
Technical Feasibility of Studying Natural Dietary Chemicals. The research proposed herein exploits recent technical and conceptual advances in biomedicine. In particular, advances in next generation sequencing technologies, epigenetic arrays, and increased performance, throughput and sensitivity of chemistry platforms such as liquid and gas chromatography now allow us to explore dietary chemical interactions with hosts at a systems biology level that was previously impossible. Recent gains in in silico technologies, including bioinformatics pipelines, reference databases and integrative statistical models for examining multiple “omics” datasets is finally beginning to catch up with our ability to generate these datasets. These hypothesis-generating advances in big data analysis combined with our extensive expertise 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) will allow us to pursue this work. Advances in genetic engineering, such as CRISPR technology now permit the targeted manipulation of genetic material and can be used to increase beneficial and reduce harmful chemicals produced by plants and microorganisms that are in the US food supply.
Using a combination of approaches, W-122 members are establishing the benefits of nutrients such as omega-3 fatty acids, fiber, and iron as well as non-nutrient phytochemicals like indoles and polyphenols. They have also been used by W-122 investigators to identify adverse effects of mycotoxins, phytoestrogens and other hormone mimics in the food supply, as well as carcinogenic and inflammation-inducing microbial products resulting from catabolism of dietary components. Availability of current technologies and the diverse expertise of W-122 researchers allows us to embrace a “field to fork” approach for ensuring a safe and health benefitting food system.
Advantages of Multi-state Study of Bioactive Dietary Chemicals. The collaborative nature of W-122 researchers provides the ideal approach to unraveling the complex role of dietary bioactive chemicals in development of cancer, metabolic diseases, and autoimmune disorders. The proposed work requires collaboration from those with diverse academic backgrounds (toxicology, molecular biology, microbiology, genomics, nutrition, food science and risk assessment) and geographic diversity due to the wide range of food crops and dietary patterns that may be involved. By approaching these nation-wide issues as a collective, we are able to bring together this diverse expertise to approach problems from various angles. Each participating research station also has unique facilities and research capabilities to ensure that we can fully address the complex issues involved in examining beneficial or detrimental effects of dietary chemicals. In addition to creating complementary approaches, W-122 collaborations limit duplication of research efforts to facilitate progress. Past collaborative efforts have been important for completing research and providing recommendations that have had far-reaching influence in terms of dietary recommendations for disease prevention and in determining acceptable levels of exposure of specific dietary toxicants. They have also resulted in development of model systems and research tools that have been implemented both by other W-122 researchers as well as the broader research community. Continued interactions between the W-122 researchers are likely to result in further progress that will influence public policy, food production and processing systems, and facilitate future research efforts. In addition, the positions of W-122 members as faculty at major land-grant universities and USDA facilities ensure that data arising from collaborative activities will be disseminated to the greatest extent possible among stakeholders and will thus provide maximum benefits to the U.S. public. W-122's efforts and focus are not duplicated in any other regional project.
Impacts of Studying Dietary Bioactive Chemicals. There are a number of positive impacts that will result from this work. First, this research will continue to improve our understanding of the mechanisms responsible for the beneficial and detrimental effects of dietary bioactive chemicals. This knowledge is the foundation for determining recommendations of dietary intakes for optimal health and disease prevention, and advancing the field of personalized nutrition which strives to provide individualized dietary recommendations based on a person’s genetics, microbiome, and other factors. Second, this research will improve the safety of the food supply by determining toxic exposure levels of adverse dietary bioactive compounds as well as identifying ways that food can be grown or processed to mitigate safety risks. Third, the discovery of novel bioactive compounds, beneficial human-associated bacteria, or development of new crop varieties as a result of this research could provide new opportunities for disease prevention or treatment. Finally, research tools developed by W-122 researchers, such as reporter cell lines, new animal models, and biomarker identification can be widely implemented to improve the quality future research in this and related fields.
Related, Current and Previous Work
W-122 researchers have made significant contributions in understanding the role of natural chemicals found in the diet on human health and food safety. Past work has focused on understanding the mechanisms by which beneficial dietary components and dietary toxins influence disease development, discovering novel bioactive components, and determining how to increase the health benefits and safety aspects of food. Some tangible outputs of this work include new tools for researchers, such as an avian model suitable for studying sensitivity to dietary carcinogens, a cell line for detecting endocrine active chemicals and a new rodent diet that more closely represents a typical US diet. They are also contributing to the development of a rating system, or toxicologic equivalency factor that can be applied to food safety assessment of fungal toxins found in food and feed and are establishing a metabolomic “fingerprint” from human biofluids and that could serve as a quantitative measure of fruit and vegetable intake, to complement or improve upon current recall-based assessments currently used in nutritional epidemiology. Finally, they have identified novel bioactive dietary chemicals and determined how processing and chemical/extract combinations influence activity. These data have been used in the formulation of new products, such as dietary supplements and anti-microbial food additives, inform growers as to specific crop varieties which may have more health beneficial properties, and how to process foods to maximize benefits and/or reduce harmful compounds.
Several recent W-122 projects have furthered our goal of determining the mechanisms of action of beneficial dietary components. Ricketts and colleagues (Nevada) have been working to elucidate the molecular mechanism of action of grape seed procyanidin extract (GSPE) in lipid regulation. They have shown that GSPE can reduce triglyceride levels elevated by a high fructose diet. GSPE inhibits hepatic lipogenesis via down-regulation of genes including, sterol regulatory element binding protein 1c (Srebp1c) and stearoyl-CoA desaturase 1 (Scd-1) in the fructose-fed animals. Through further experimentation, they determined that GSPE selectively regulates intestinal FXR-target gene expression in vivo, and modulation of bile acid absorption and transport is a critical regulatory point for the consequential hypotriglyceridemic effects. Combination with a bile acid sequestrant, chylestyramine (CHY), reduced serum triglycerides by 25% more than CHY alone, suggesting GSPE provides additive and complementary efficacy as a lipid-lowering combination therapy in conjunction with CHY.
At Michigan State University, the team of James Pestka has a model that enables dissection of the countervailing roles of silica (potentiation) and DHA (attenuation) in lupus initiation. Lupus is a debilitating autoimmune disease that affects 1.5 million Americans. While the genome is a predisposing factor for autoimmunity, lifetime exposures to factors (i.e., exposome) such as environmental toxicants and diet are now recognized to modulate hereditary effects. Experimental animal and epidemiological studies have linked exposure to the respiratory toxicant silica to lupus.
Several W-122 researchers are determining mechanistic links between the composition of the gut microbiota and microbial metabolites with human disease such as cancer and cardiometabolic diseases. Abby Benninghoff (Utah) has demonstrated the effects of anthocyanin-rich tart cherry powder on development of colitis-associated colorectal cancer is dependent on the background diet of the host. Mice fed an optimized diet were protected while those on their total western diet formula were not.
Nerurkar (HI) and Weir (CO) have also explored effects on the gut microbiota as a mechanism for anti-diabetic effects seen in mice fed a high fat diet in conjunction with various preparations of noni juice. Weir’s group has also examined the influence of consuming stabilized rice bran and navy bean powder on altering gut microbial profiles and modulating microbial metabolites in colorectal cancer survivors. They found that consumption of stabilized rice bran increases populations of mucin-degrading bacteria that might facilitate the increased turn-over of intestinal mucins and reduce exposure to pathogenic organisms and luminal toxins. Zhu (WSU) determined that Goji-berries, a polyphenol rich food traditionally used in Chinese medicine was protective against DSS-induced colitis in mice. Turner (TX) has also explored polyphenol-rich fiber sources, such as dried plum, reduced colon cancer in carcinogen-injected rats. Compounds produced by microbial metabolism of plum phenolics in the lumin of the rat colons have been demonstrated to protect against carcinogenesis.
Turner (TX) is also examining the effects of the intestinal metabolome, created by catabolism of dietary compounds by gut microbiota, on gene regulation via epigenetic mechanisms including histone acetylation and DNA promoter methylation. Altered patterns of gene expression in the host induced by changes to the epigenome are critical in the regulation of cell proliferation, differentiation and apoptosis, as well as short chain fatty acid transport. The benefits derived from these compounds are not constrained to the colon, because they have also shown benefits to bone health as well.
Interactions between diet and host microbiome are emerging as important factors in the development of many diseases. As such, understanding how dietary components are metabolized by gut microbiota, how diet changes the composition and production of important microbial short chain fatty acids, and what the downstream effects are on mucin production, immune responses, gut barrier function, and intestinal inflammation are becoming increasingly important in preventive medicine. W-122 researchers have already made important contributions and are continuing to make headway in this area. As a result, we have made investigation of effects of phytochemicals and other dietary components on gut microbiota and intestinal function a separate objective for this renewal application.
In addition to identifying mechanisms and molecular targets for beneficial natural chemicals, W-122 researchers have been elucidating mechanisms of dietary toxicants and developing biomarkers for risk assessment and disease prevention. These compounds include microbial toxins, such as mycotoxins, that commonly enter the food supply, by-products of food processing (ie. acrylamide), and certain nutrients that are over-consumed in a western diet, such as saturated fats, processed sugars and fructose. Research by W-122 members have resulted in a better understanding on how these compounds effect individuals and their subsequent generations, but have also led to the development of novel model systems that have facilitated these studies.
Mycotoxins ahre fungal metabolites that are typically associated with crops and represent a threat to human and animal health. Deoxynivalenol (DON or "vomitoxin"), is a trichothecene mycotoxin produced by Fusarium and often found in cereal grains. Climate change, modified agricultural practices and recent trade globalization have increased Fusarium cereal blight, thereby increasing grain DON contamination and expanding the contaminant profile to include other structurally-related 8-ketotrichothecenes (3-ADON, 15-ADON, nivalenol, fusarenon X) as well as plant glucosidic metabolites such as DON-3-glucoside. Existing data are insufficient to predict the toxicity risks from exposure to mixtures of these emerging trichothecenes. James Pesta (MSU) is testing the hypothesis that toxic equivalency factors (TEFs) for the 8-ketotrichothecenes derived from DON toxicity models will be applicable to food safety risk assessment and toxicity analysis. Currently they are applying this knowledge by developing a simple in vitro assay that will enable measurement of the trichothecene toxic equivalents in food samples.
Coulombe and colleagues (UT) have developed an avian embryo exposure model that reduces use of live animals in research, and allows them to study the molecular mechanisms regulating the universal detoxifying enzyme glutathione S-transferases. The previously demonstrated that domesticated turkeys (Meleagris gallopavo) are sensitive to aflatoxicosis, while Eastern wild turkeys (M. g. silvestris) are relatively resistant. Changes in methylation, particularly at CpG sites clustered in the promoter region of a gene, can lead to changes in expression. To identify whether methylation differences may be responsible for GST silencing in domesticated turkeys, Coulombe’s group has designed primers to amplify the GSTA4 promoter region and will be using a methylation sensitive restriction enzyme-based method to determine whether the CpG sites are differentially methylated.
Another type of dietary carcinogens are polycyclic aromatic hydrocarbons. Williams’ group (OR) has conducted a human clinical trial to determine the pharmacokinetics of PAH exposure equal to levels found in food. They have also determined in a rodent model that PAHs transfer to the fetus, and increase cancer incidence. However, indole-3-carbinol from crucifers, when fed to pregnant mothers, protects the offspring via epigenetic alterations.
Overexposure to some dietary nutrients, such as certain fats, can compromise human health. Romagnolo (AZ) is exploring molecular mechanisms of dietary fatty acid exposure on development of inflammatory bowel diseases and colorectal cancer. Eisenstein (WI) is exploring mechanistic parallels between excess dietary fat and excess uptake of dietary iron in obesity and iron overload. Benninghoff’s group (UT) has shown that consuming a total western diet (high fat and high sugar) has multi-generational effects on colorectal cancer development in a rodent model and that ancestral exposure to this dietary pattern may be as detrimental as direct exposure to the diet. They are currently exploring beneficial compounds, such as green tea extracts, to mitigate these effects.
W-122 researchers are also exploring ways in which food processing may reduce negative health impacts or increase beneficial impacts as well as ways to use natural chemicals to improve food safety in packaging and processing. Helfrich and colleagues (IL) have studied the impact of soy isoflavones in breast cancer development or protection. A key aspect of their work is that the form in which these compounds are consumed strongly influences their biological activity with regard to breast cancer. They found that a soy flour diet prevented isoflavones from stimulating MCF-7 tumor growth in athymic nude mice, indicating that other bioactives in soy can negate the estrogenic properties of isoflavones. An examination of global gene expression profiles of ovariectomized animals fed either the soy flour diet or purified isoflavones suggest that dietary soy flour affects gene expression differently than purified isoflavones, which may explain why soy foods prevent isoflavone-induced stimulation of MCF-7 tumor growth in athymic nude mice. Helfrich and colleagues are also examining how thermal abuse of various types of cooking oils influence their ability to cause intestinal inflammation in collaboration with Benninghoff (UT).
Friedman and colleagues (USDA_CA) have demonstrated that combinations of shiitake mushroom (Lentinus edodes) mycelia with either fermented elm tree (Ulmus parvifolia) bark extract or turmeric are protective against several disease states. In the first study, they found the combination including elm tree bark extract resulted in anti-inflammatory and anti-allergic actions in a mouse model of allergic asthma. In the second study, they found the combination including turmeric protected mice against both liver damage (necrosis) and lethality induced by Salmonella Typhimurium. The protection was induced by stimulation of the immune system. Nerurkar (HI) and Weir (CO) have also examined the effects of fermentation on various preparations of noni juice. Global metabolite profiles showed that fermentation was the major variable driving differences in chemical composition of various noni preparations, but fermented juices did not confer specific benefits in a rodent model of diabetes and metabolic syndrome.
Processing plant foods at elevated temperatures forms acrylamide, a potential carcinogen. A wide range in the acrylamide concentrations was found in commercial canned black ripe olives. Freidman (USDA_CA) has optimized a safe thermal processing condition that is an efficient alternative to current approaches, and reduces acrylamide formation during thermal processing of black ripe olives. They have also explored ways to minimize microbial food contaminants that pose potential food safety issues. They have found the glycoalkaloid tomatine in tomatoes exhibits potent antimicrobial activity against pathogenic protozoa (Trichomonas vaginalis) that can infect humans, cattle, and cats, suggesting its potential as an alternative therapy for trichomoniasis. They have also found that a commercial pomegranate preparation reduced the heat resistance of the virulent pathogen Escherichia coli O104:H4 in ground chicken, suggesting the described kinetic model can be used to design lethality treatments to reduce pathogenic E. coli at lower processing temperatures. Zhu (WA) has also examined methods of inhibiting growth of E.coli O157:H7. Treatment of this pathogen is difficult since many antibiotics induce SOS response and enhance Stx production, requiring alternative antimicrobial interventions. They found that Cinnamon oil effectively inhibited growth of E. coli O157:H7 at high concentration and reduced both Stx2 phage and total phage induction at a sub-inhibitory concentration. Inhibition may be due to suppression of bacterial SOS response regulator RecA. Additionally, C. cassia oil had bactericidal effects against bovine mastitis pathogenic isolates possibly through disruption of membrane structure.
Freidman (CA-USDA) has also explored the impact of several plant-based antimicrobial washes on the sensory properties of celery, lettuce, and spinach. Greens treated with 0.1% cinnamaldehyde had the highest taste preference by panelists, suggesting its value for large-scale use to improve microbial food safety.
Research by W-122 researchers has successfully met the objectives of improving human health and food safety through a better understanding of the mechanisms of action of natural products in health and disease and through discovery of novel compounds/extracts that can reduce food-borne pathogens. The objectives in the current application represent the natural expansion of this work and stem from collaborative efforts of project members. In particular, modulation of gut health and epigenetic regulation of gene expression represent new directions highlighted in the current objectives. Understanding mechanisms of action of natural products and determining how processing effects the bioactivity and safety of natural chemicals will remain an emphasis.
Examine the effects of phytochemicals and other dietary components on gut microbiota and intestinal function.
Comments: Emerging research has indicated that the intestinal milieu, including the gut microbiota and it's metabolic capacity, may be at the root of many chronic diseases. W4122 researchers will explore how dietary components influence the gut microbiota and intestinal environment to influence health outcomes using a combination of cell and animal models and human feeding trials.
Identify cellular mechanisms and host molecular targets of beneficial or adverse dietary components that influence human health.
Comments: Although we know that dietary components can positively or negatively impact human health, the cellular basis of nutrient-host interactions are often poorly understood. W4122 researchers, using model systems, will examine the mechanistic interactions by which specific nutrients and other dietary components interact with host cells.
Explore the interaction between dietary components and the host metabolome and epigenome.
Comments: Metabolites represent a terminal product of the central dogma. Therefore, determining changes in global metabolite profiles can provide mechanistic insight into the influence of dietary perturbations on host physiology. Metabolite profiling can also be used to identify biomarkers of dietary intake or host health status and can indicate how various processing procedures affect the quality of foods. Epigenetics represent a heritable but modifiable means of regulating gene expression. Understanding epigenetic responses to dietary components and food borne toxicants will provide key insights into inter-individual differences in response to dietary components and offers new targets for therapeutic interventions.
Determine how food processing influences chemical composition to affect human health.
Comments: Although many important bioactive dietary components have been identified and characterized for their impacts on human health, common food processing practices can alter the effects of these components. W4122 researchers will explore how common food processing practices alter food chemistry and subsequent impacts on human health. They are also exploring value-added uses of food wastes that may contain bioactive components important in human and animal health and food safety.
Introduction. Through years of interactions, coordination of research efforts and active collaborations, PIs at different stations in the region have developed and maintained expertise and facilities in specific areas which complement and contribute to each other. When these various research capabilities are shared through active collaborations, they greatly enhance and synergize the research productivity of W-122 scientists. Several of the major active and planned collaborative studies among W-122 scientists have been summarized (Table 1), along with the special contributing research capabilities of different Stations (Table 2). All data will be shared among all project members and summarized activities will be accessible to the public via a W-122 web site.
Objective 1: Examine the effects of phytochemicals and other dietary components on gut microbiota and intestinal function. The following areas are proposed for further research:
a) AZ plans to investigate the effects of high fat diet on expression of markers of inflammation and bile acid receptors in intestinal and liver cells. We will conduct both cell culture and animal studies that will include transgenic mouse models of the farnesoid X receptor (FXR) and study how interactions between loss of expression of adenomatous polyposis Coli (APC) and changes in FXR expression influence cell proliferation, inflammation, and production of secondary bile acids.
b) CSU will use Illumina sequencing and a combination of human, animal, and in vitro models to assess the impact of various dietary components on the gut microbiota and intestinal health and how intestinal inflammation and gut dysbiosis drive downstream pathologies such as colorectal cancer, endothelial dysfunction and other cardiometabolic manifestations. In particular, we are in the process of identifying how western-style diets drive endothelial dysfunction via dysbiosis of the gut. Using a series of antibiotic treatments and microbiota transplants in mice displaying endothelial dysfunction, we plan to identify specific bacteria that are either beneficial or detrimental to cardiometabolic health. In addition, we are exploring how polyphenols in a Chinese fermented tea can modulate the intestinal environment to protect against inflammation-induced endothelial dysfunction using a DSS-induced mouse model of chronic colitis.
Insufficient consumption of dietary fiber can result in degradation of the intestinal mucus layer, as bacterial “grazers” switch from metabolizing these dietary fibers to consuming the intestinal mucins. We are planning to use rodent and cell culture models to explore the role of prebiotic fiber components, such as arabinoxylan from rice and wheat, in maintenance of homeostasis of the intestinal mucus lining and modulation of mucin-degrading bacterial populations.
Finally, we are examining the effect of bacteria phage consumption as a form of “prebiotic” in a human population. Adult individuals that experience mild to moderate gastrointestinal issues will consume either a phage-filled capsule or a placebo control for 1 month in a double blinded crossover study. Study endpoints include basic safety profile of phage consumption (comprehensive metabolic panels), changes in GI symptoms as determined from a previously validated IBS questionnaire, gut microbiota changes, and measures of GI and systemic inflammation.
c) HI plans to discover and characterize novel bioactive compounds or chemical fingerprints that have beneficial or adverse effects on human health by modulating gut microbiome. In particular, we will collaborate with CO to explore how noni juice, a traditional Polynesian medicinal preparation, bitter melon, and coffee affect gut microbial composition and parameters related to insulin sensitivity and metabolic syndrome in mice. In addition, we will look at how coffee consumption affects gut microbiota composition and health-related parameters in a human population.
d) OSU will determine the impact of the microbiome on the synthesis of endogenous ligands for the aryl hydrocarbon receptor (AhR) and subsequent impact on the response to dietary carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) that are thought to act via AhR signaling and metabolized by AhR regulated genes. In addition, we will explore the impact of the microbiome on levels of trimethylamine-N-oxide (TMANO) and interaction with genetic variants of the human liver enzyme, flavin-containing monooxygenase 3, responsible for conversion of trimethylamine (from the breakdown of dietary choline and from consumption of fish) to TMANO. The levels of TMANO have been shown to have a marked influence on cardio-vascular disease. OSU is further interested to understand the role of diet and inflammation on the lung mucosal microbiome, which similar to the gut microbiome may play an important role in host response to inflammatory-related diseases. We propose to evaluate the importance of diet on the lung microbiome in mice linked to specific markers of inflammation.
e) TAMU will explore how various dietary components rich in polyphenols and fiber promote chemoprotection in models of chemically-induced colon carcinogenesis through maintenance of a healthy gut microbiota and generation of beneficial metabolites as well as epigenetic regulation of colonocyte gene expression. We will quantify colonocyte proliferation and apotosis in a rat model of chemically induced carcinogenesis as well as characterizing expression profiles of Toll-like receptors, SCFA transporters, and immune and metabolic regulators. We will also characterize microbiota changes over time and nutrient transport throughout the GI tract and epigenetic alteration of the DNA in colonocytes.
f) UT plans to determine the impact of dietary supplementation with functional foods rich in bioactive polyphenols, including BRB, on colon tumorigenesis using the azoxymethane/dextran sodium sulfate mouse model of colitis-associated colorectal cancer (CAC) and a rodent basal diet that reflects typical US nutrient intakes (the total Western diet). Changes in the gut bacteria associated with inflammation and tumorigenesis directly contribute to colon tumorigenesis, and dietary interventions with bioactives known to modulate the gut microbiome and reduce gut inflammation may contribute to reduced incidence or severity of CAC. Specifically, we will 1) Determine the efficacy of black raspberries for suppression of colon tumorigenesis using a pre-clinical mouse model of CAC and 2) assess the effects of dietary interventions on composition of the gut bacteria community and capacity for BRB-conditioned bacteria to protect the host against CAC following fecal transfer. Our experimental approach takes into account the dynamic response of the gut microbiome, as well as the dynamic response of the gut epithelium, to intervention with bioactive food components during active colitis, a period of recovery and (if failed recovery) progression to colitis-associated carcinogenesis.
g) WSU plans to examine how dietary fiber and polyphenols in grape pomace modulate epithelial cell differentiation intestinal inflammation, potentially via CDX2. We will conduct both animal and cell culture studies. For animal studies, we will use both wild-type and transgenic mice which will be fed a diet containing various levels of grape pomace to elucidate the biological effects of fruit on gut epithelial differentiation and barrier function. To establish the causal relationship, we will also conduct cell culture studies. The advantages of cell culture studies include the ease to inhibit or activate, to knockdown or over-expression of key signaling mediators, fitting well for mechanistic oriented studies. Based on data obtained from animal and cell culture studies, we also plan to develop functional foods maximizing the beneficial effects of dietary factors on gut health.
Objective 2: Identify cellular mechanisms and host molecular targets of beneficial or adverse dietary components that influence human health. The following issues are proposed for further research:
a) AZ will investigate genomic interactions of bioactive phytochemicals with disease pathways (proliferation, apoptosis, and autophagy) to elucidate molecular mechanisms related to disease prevention.
b) HI will conduct studies to explore the role of functional foods (coffee, bitter melon, and noni) in modulating microRNA in mice and humans.
c) IL has developed utilizes a model murine metastatic mammary carcinoma 4T1 cells in BALB/c mice. In this model we utilize a transplantable tumor cell line that grows aggressively as tumors when implanted or injected into the tibial marrow cavity of BALB/c mice. This allows us to determine the effect of various dietary components on BC progression. Because the tumors grow in animals that are not immune compromised and readily metastasize, the 4T1 tumors are a valuable pre-clinical model for analysis of breast tumor growth in bone and subsequent metastasis to visceral tissues, and they are appropriate for studying the impact of dietary components on these processes. These cells are engineered to constitutively express luciferase; so their distribution in vivo can be readily followed, non-invasively and in real-time, by BLI. This feature provides major advantages in terms of sensitivity and economy in animal use, because time course studies can be conducted on single animals, with necropsy performed at an optimal time. Thus, we believe that our 4T1 mammary cancer model is an appropriate and convenient preclinical model for studying BC metastasis and its regulation by dietary components.
d) MSU will explore the mechanisms by which n-3 polyunsaturated fatty acids suppress environmentally-induced autoimmune diseases, using lupus as a model AI disorder. Research studies reveal that consumption of n-3 polyunsaturated fatty acids (PUFAs) found in fish oil holds promise for preventing and ameliorating chronic inflammatory diseases including autoimmune nephritis. We will test the hypothesis that consumption of n-3 PUFAs will suppress silica-accelerated nephritis in lupus-prone mice and that this will correspond with decreased leukocyte recruitment and inflammation-associated gene expression in the kidney.
e) OSU is partnering with Lawrence Livermore National Laboratory to assess the absorption, metabolism and excretion of PAHs following oral exposure. This is made possible by a recent development of a UHPLC-accelerator mass spectrometry assay that can measure [14C]-benzo[a]pyrene (BaP) and its metabolites in plasma and urine in the low femtograms (10-15 grams)/mL levels. We have demonstrated to FDA (FDA IND 117175) that the micro-dose (46 ng of BaP and 5 nCi of [14C] represents a de minimus risk to volunteers. We are also investigating how genetic variants of enzymes responsible for BaP metabolism alter the metabolism and excretion of BaP after oral dosing. Future studies will assess the potential of Brussels sprouts consumption on BaP pharmacokinetics. The goal is to provide better risk assessment data for regulatory agencies, identify the most susceptible individuals and gauge the potential for diets high in cruciferous vegetables to provide protection. To further improve risk assessment of PAHs, we propose to identify pathways and molecular targets predictive of carcinogenic potential of PAH chemicals alone and in complex mixtures consumed by humans using signatures derived from global sequencing and metabolic data.
f) Rutgers will examine the preventative actions of fruit extracts of red raspberry (Rubus idaeus) and related polyphenols on the development of diet induced obesity (DIO). The central hypothesis is that raspberry derived phenolic compounds have a dual action to suppress feeding and to increase fat oxidation. This proposal will use diet-induced obese (DIO) mice to investigate the acute and long-term effects of raspberry extracts on gastrointestinal physiology and interaction with hindbrain and forebrain neural targets. In addition, we will determine the bioacessibility of raspberry extracts using a functional model of the human digestive system (TNO intestinal model) and in vivo animal model to determine the metabolism and bioavailability of raspberry derived phenolic compounds. The findings from these studies will identify a metabolic and molecular signature of raspberry-derived compounds on feeding and metabolic outcomes to impact human health.
g) UCD will examine molecular mechanisms by which a variety of dietary components, chemicals and extracts can affect the growth and proliferation of and biochemical responses in liver, breast and intestinal cancer cells. Analysis of the ability of these dietary materials to specifically interact with and activate/inhibit the aryl hydrocarbon receptor (AhR) and estrogen receptors (ERa and ERb) (key ligand-dependent regulatory factors affecting cell proliferation and cellular and immunological responses) will be determined using a combination of in vitro and recombinant cell-based bioassays. The direct interaction of the chemicals/extracts with the receptors will be examined using ligand- and DNA-binding and receptor-dependent gene expression analysis. Extremely high levels of chemicals with ER/AhR activity have recently been identified in extracts of a variety of common food products and we will utilize bioassay directed fractionation and instrumental analysis approaches to isolate and identify the responsible chemical(s), and will characterize the relative potency and efficacy of these dietary components on receptor-dependent biological and cellular responsese.
h) UT-RC will continue to examine the molecular mechanisms underlying extreme sensitivity to dietary carcinogens such as Aflatoxin B1 utilizing a previously developed avian model. Our avian model exhibits silencing in critical cancer-protective gene glutathione S-transferase (GST), similar to susceptible human populations. We will examine possible genetic and epigenetic mechanisms for GST regulation, the role of GSTs in cancer protection and chemoprevention, and comparing related avian species in these processes. To explore the mechanisms underlying GST silencing and sensitivity to dietary carcinogens, we will perform miRNA sequencing in avian liver, and conduct antibody-based pull-down assays to evaluate the functional role of candidate miRNAs discovered and assess the possible consequences of gene splice variants in GST gene function in wild and domestic birds.We will also expand on the current model by developing, validating and optimizing an isolated avian hepatocyte model system to explore the function Nrf2/Keap1 transcription factors in real-time.
i) UT-AB will determine the molecular responses of colon epithelial cells to dietary interventions (the total Western diet with and without supplementation with black raspberries) by measuring perturbations in pathways regulating inflammation and cell cycle progression. Anthocyanidins (the aglycone form of anthocyanins), such as cyanidin and delphinidin, modulate a variety of cell signaling pathways involved in inflammation, carcinogenesis and angiogenesis, including suppression of expression and/or signaling through COX-1 and -2, iNOS, Akt, ERK1/2, TNFα, NFκB, IL-6 and IL-8. Gene expression will be determined using the Fluidigm BioMark HD 96x96 microfluidics system, wherein expression of 96 genes is measured in up to 96 independent samples. Candidate genes (n=96) that are known to be critical for CAC have been selected for the Fluidigm chip, including transcripts involved in inflammation, regulation of cytokines, immune response, cell cycle regulation, apoptosis, etc.
j) WI will explore mechanisms of dietary iron absorption underlying obesity-associated anemia. We will determine the impact of iron regulatory protein 1 (IRP1) in controlling dietary iron absorption in mice and to determine if IRP1 control of hypoxic inducable factor 2alpha (HIF2alpha) is important in the control of dietary iron absorption in neonatal swine. IRP1 is the key iron mediator of HIF2alpha mRNA translation. IRP1 is a repressor of HIF2alpha synthesis and loss of IRP1 translationally activates HIF2alpha mRNA. HIF2alpha is a transcriptional activator of the main blood forming hormone erythropoietin and also the iron transport system controlling dietary iron acquisition in the duodenum. Mice lacking IRP1 develop a severe but transient polycythemia (too many red cells) and absorb more iron to promote red cell overproduction. We will also continue our studies on the role of IRP1 in controlling erythropoiesis.
Objective 3. Explore the interaction between dietary components and the host metabolome and epigenome.The following issues are proposed for further research:
a) AZ will investigate the epigenetic regulation of breast cancer susceptibility genes in cell culture and animal models. Specific focus will be on dietary regulation of the BRCA-1 gene in mammary epithelial cells and mechanisms of epigenetic regulation involving DNA methylation and histone modification.s associated with the BRCA-1 gene, and expression of non-coding RNA that interfere with normal regulation of BRCA-1.
b) CSU will examine metabolite profiles of human biofluids, including urine, plasma, and stool to identify biomarkers that correspond to dietary intake levels of fruits and vegetables. Fruit and vegetable consumption has been inversely correlated with risk of cancer, cardiometabolic diseases, and all cause mortality but it is difficult to determine the exact protective mechanisms and doses required for beneficial effects. This is due, in part, to a lack of reliable and quantifiable fruit and vegetable intake data in large nutritional epidemiology studies. We are exploring traditional global metabolomic platforms, ionomics, and pioneering a novel metabolomics approach called Indiscriminate Mass Spec/Mass Spec Phytochemical Substructures (IMPS) to identify quantifiable and reproducible metabolomic signatures associated with fruit and vegetable intake.
c) HI will examine the metabolome profiles of functional foods such as coffee, bitter melon and noni juice to identify novel bioactive compounds that may influence host metabolic status and insulin sensitivity.
d) OSU is utilizing a transplacental model of cancer induction by dietary PAHs and chemoprevention by dietary components of cruciferous vegetables, primarily indole-3-carbinol and sulforaphane. The focus of this research is understanding how maternal exposure to dietary carcinogens capable of crossing the placenta and inducing cancer in offspring alter the epigenome. Currently, we are examining miRNA and lncRNA profiles in target tissues of neonatal offspring born to mice treated with PAHs during pregnancy and fed diets containing cruciferous vegetables. We are also evaluating the role for miRNAs in regulating pulmonary inflammation and disease in response to a high fat diet, which changes the basal transcriptome and proteome of the lung and alters susceptibility to inflammation after insult.
e) TAMU will determine whether radiation-induced colon disease is a result of perturbation of the epigenetic signature of colon stem cells and whether intervention with dietary bioactives can mitigate these effects. We hypothesize that low dose HZE radiation-induced apoptosis avoidance and colon tumor formation is the result of perturbations in the expression and activity of epigenetic effectors that can be mitigated with dietary countermeasures. Specifically, we propose that adult stem cells residing in mouse colon crypts are highly sensitive to radiation-induced perturbations to the epigenome, and that the resulting epigenetic “signatures” will be transferred to daughter cells rendering them resistant to apoptosis induction. Our goal is to determine the impact of low dose HZE ions on perturbations in intestinal whole genome methylation and histone acetylation status. Additionally, we will determine whether colon adult stem cells are more susceptible to epigenetic perturbations induced by low dose HZE radiation than non-stem cells in the same colon crypt. Finally, we will examine potential dietary countermeasures that alter colonic microbiota and butyrate concentrations in mitigating the impact of low dose HZE radiation on epigenetic regulation of apoptosis in colon adult stem cells and daughter cells.
f) UT-RC will determine the role of gene methylation in GST expression and silencing in the GST null phenotype of their avian model.
g) UT-AB will assess differential DNA methylation for promoter regions of candidate genes involved in colon carcinogenesis in colon tissues of F3 generation offspring exposed directly, ancestrally via F0 generation grandparents or over multiple generations (F0 through F3) to the total Western diet (TWD) as compared to mice fed either a standard basal diet (AIN93G) or a simple high fat diet (45% fat DIO). Incidence of CAC was significantly higher in F3 offspring exposed ancestrally to the TWD as compared to their F3 counterparts fed AIN93G (56%). Moreover, tumor burden was markedly higher (> 3-fold increase) in F3 offspring exposed to TWD over multiple generations when compared to F3 offspring provided TWD directly.
Objective 4. Determine how food processing influences chemical composition to affect human health.The following issues are proposed for further research:
a) CA-USDA, in collaborative studies with Dr. Wallace Yokoyama (WRRC, ARS, USDA, Albany, CA), will evaluate anti-obesity properties of food processing byproducts (wastes) such as freeze-dried apple, orange, and potato peel powders as well as mushroom processing byproducts in mice on a high-fat diet and relate bioactive compound composition to efficacy. In addition, we will evaluate the inactivation of antibiotic-susceptible and antibiotic-resistant strains of Trichomonas vaginalis protozoa that cause disease in humans, cattle, and cats using natural plant-based pure compounds and plant extracts in collaborative with Professor Kirkwood Land of the Department of Biology, University of the Pacific, Stockton, CA.
b) HI will examine the differences in chemical composition and anti-obesity effects of fermented an non-fermented preparations of noni juice as well as explore the differences on physiological parameters in humans consuming different coffee roasts.
c) IL will investigate the effect of processing and cooking on altering frying oil. We have fried fish, chicken and potatoes for multiple frying cycles (up to 100 cycles) to generate thermally abused frying oil (TAFO). We are currently using the BC metastasis model described in Objective 2 to evaluate the effect of TAFO on metastatic progression.
d) UT-AB, in a collaboration with IL, proposes to investigate the impact of thermal abuse of cooking oil on gut inflammation, composition of the gut microbiome and development of inflammation-associated colorectal cancer in mice fed a standard basal diet (AIN93G) or the total Western diet. Diets will be prepared using soybean oil that has not been abused or oil that was abused by repeated high temperature thermal cycling while cooking fish. The azoxymethane/dextran sodium sulfate model of inflammation-associated colorectal carcinogenesis will be employed using male C57BL/6J mice. Endpoints assessed will include food/energy intake, body weight gain, body composition, colitis in response to dextran sodium sulfate, colon tumorigenesis, biomarkers of inflammation (Fluidigm PCR as described above in 2), and assessment of the gut microbiome (taxonomy, alpha and beta diversity and metagenome).
Measurement of Progress and Results
- Evidence-based information on the beneficial and adverse impacts of bioactive, dietary chemicals on human health and chronic disease for consumers and clinicians.
- Improved recommendations for use, standardization and processing of bioactive dietary compounds in herbal supplements and functional foods.
- Improved hazard and risk assessment data of dietary toxicants for policy makers.
- Identification of novel value added crops and foods that can be exploited by farmers and processors, respectively.
- Data on the effects of various dietary components on gut health that can be used by dieticians and other clinicians.
- Identification of new packaging/processing methods to reduce the risk of food-borne toxicants and microorganisms.
- Dissemination of data in scientific meetings, extension publications, and peer-reviewed journals.
- Creation and archiving of resources and data such as purified natural chemicals, extracts with biological activity, biological reagents such as antibodies, cell lines, RNA from treated cells, laboratory animals, and big datasets generated from “omics” technologies to be made available for further study by W-122 participants and other researchers.
Outcomes or Projected Impacts
- Safer and more efficacious dietary supplements and functional foods. The work of W-122 researchers in determining mechanisms of natural chemicals derived from foods or micro-organisms will help in determining specific beneficial compounds or combinations and doses needed to activate molecular targets. Research outputs will also be useful in determining manufacturing processes and delivery mechanisms that increase efficacy and improve safety.
- Reduce the health burden of chronic diseases such as cancer and metabolic syndrome. Information from studies conducted by W-122 researchers will help identify dietary chemicals and whole foods that are efficacious in preventing or reversing chronic diseases by identifying beneficial dietary chemicals that that confer health benefits to the host such as, preventing development of colonic lesions and improving gut barrier function, increasing insulin sensitivity, and modulating inflammation.
- Improved dietary recommendations for optimizing health throughout the lifespan. W-122 work in areas such as diet-microbiome and epigenetic effects of dietary components will help inform how dietary recommendations can be tailored for improved outcomes related to maternal-child health, healthy aging, and other milestone throughout the lifespan.
- Development of new tools for nutritional research and epidemiological studies. W-122 researchers are developing cutting edge model systems, nutritionally relevant animal diets, and identifying biomarkers of dietary intake and disease that will be valuable tools for nutrition researchers, educators, and possible clinical applications.
Milestones(0):See attached Powerpoint.
Projected ParticipationView Appendix E: Participation
New information and conceptual insights resulting from this research will be communicated to a variety of stakeholder groups through varying media. Research details will be published for distribution in peer-reviewed scientific journals and translational findings will be communicated through extension literature. Findings will also be incorporated into teaching modules for K-12 education and integrated into undergraduate and graduate curricula. Several W-122 researchers have already developed university-level classes that highlight the role of natural chemicals present in the diet and prodiuced by microorganisms. Lectures, webinars and online certification offerings will also be available to groups of professionals such as registered dieticians, clinical laboratory scientists, and other medical professionals as continuing education opportunities. Findings that are considered important for public distribution will be placed into a format that is suitable for dissemination through the media. When our level of understanding of new findings and their significance so dictate, the information can become the focus of workshops and training sessions for Cooperative Extension Specialists. W-122 will also establish a website and social media pages for rapid distribution of important information and for communication of constituents with W-122 members.
W-122 investigators work closely with community information organizations to provide perspectives on the possible role of diet in disease. Contributing Experiment Stations have developed effective means of communicating with poor and minority populations through cooperation with community organizations and food distribution and nutrition educattion programs, such as 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 grower's associations for which W-122 findings will be useful.
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.
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