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.