Duration: 10/01/2013 to 09/30/2018

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Diet, including specific nutrients and bioactive food components, plays a vital role in improving health and reducing risk for developmental and chronic disease. This collaborative research project brings together scientists with broad nutrition science expertise and experience to test hypotheses that ultimately will enhance the well-being of the population through reduced risk and incidence of several chronic diseases including cancer, obesity and osteoporosis. We plan to advance the understanding of the requirements for nutrients and phytochemicals in maintaining optimal health and to translate our findings to public health practice. There are numerous chronic diseases and developmental disorders for which risk may be modified in relation to exposure to bioactive dietary constituents. However, for the purpose of this collaborative research project the investigators are targeting cancer, bone health, obesity, birth defects and cognitive function and neurological disease. These diseases, and the associated interactions with nutrients and phytochemicals, are complex and require diverse approaches to advance knowledge that will lead to effective preventive and therapeutic strategies. Importantly, both the factors determining the absorption/bioavailability and targeted mechanism of actions will be addressed in this multi-state effort. A major outcome of this approach is scientifically sound dietary recommendations for communities and individuals.

The numbers are staggering when one reviews disease incidence for these chronic diseases in the United States. Cancer is the second leading cause of death in the U.S. with 1 in 2 Americans receiving a cancer diagnosis in their lifetime and over 550,000 cases diagnosed annually. Specific to our efforts, breast cancer accounts for over 180,000 cancer cases annually and prostate cancer accounts for 9% of all cancer diagnosed in males. Numerous nutrients and phytochemicals have been shown to increase or decrease cancer risk. Currently, our investigative research team is collaborating on several dietary constituents including, but not limited to, folate, biotin, zinc, vitamin E, isothiocyanates, polyphenols, soy isoflavones, lycopene and citrus monoterpenes. All of which have important roles in human health and protection against chronic disease.

Osteoporosis, another leading chronic illness in older Americans, has a prevalence rate in people over age 50 years of over 12,000,000 cases (2002) is expected to increase to an estimated 14 million cases by 2020. Consequences of osteoporosis are severe with a mortality rate of 24% in the 12 months following newly diagnosed hip fracture. We will continue to expand research on calcium and isoflavones and will expand into other nutrients and phytochemicals including vitamin D and non-digestible carbohydrates (i.e., fructooligosaccharides). Application of new and novel techniques and approaches to assess the impact of dietary interventions on bone outcomes through the collaborative and complementary expertise within the research group will afford an unprecedented opportunity for research in this area.

Perhaps no chronic illness is of greater concern to the American public than obesity. Obesity and related diseases, with cardiovascular disease and diabetes at the forefront, pose a substantial health risk for U.S. citizens and are an enormous economic burden for the health-care system (Centers for Disease Control and Prevention 2011a, 2011b, 2011c). Amazingly, the prevalence of obesity (defined as a body mass index > or = 30) is 20 to 30 percent among U.S. adults, depending on geographic region. In some regions, 10% or more of adults age 20 years and older are diagnosed with type II diabetes, and 10% or more of adults are hospitalized each year because of heart disease. Diabetes is a significant predictor of lost productivity in the U.S. workforce (Vijan et al., 2004), with the incremental lost income due to diabetes by 1992 estimated at $60 billion over an average diabetes duration of 9.7 years. From 1992 to 2000, diabetes was responsible for $4.4 billion in lost income due to early retirement, $500 million due to increased sick days, $31.7 billion due to disability, and $22 billion in lost income due to premature mortality, for a total of $58.6 billion in lost productivity. Cardiovascular disease and sleep associated pathology are also devastating to health and economic wellbeing. Inadequate sleep duration or sleep quality and obstructive sleep apnea also increase risk for obesity and cardiovascular disease in multiple populations (Lui and Sau-Man 2012, Lucassen et al., 2012). In pregnant women, cardiovascular disease causes a dramatic increase in the risk for neonatal complications (Siu et al., 1997), with complications occurring in about 17 percent of full-term pregnancies. Complications include death, respiratory distress syndrome, intraventricular hemorrhage, premature birth, and small-for-gestational-age birth weight. Efforts to reduce the clinical consequences of obesity through dietary and behavioral interventions are not only warranted, but provide significant promise. Novel approaches including green tea polyphenols, grape anthocyanins, and/or vitamin E supplementation may help to control the undesirable biological changes - including inflammation and oxidative stress responses - that further impair the health status of obese individuals.

Neural tube defects (NTD), including spina bifida and anencephalus, are diagnosed in nearly 30 per 100,000 live births in the U.S. It is well established that increased folate during early pregnancy will reduce birth defects by over 70%; however, the mechanism for protection, which is believed to be genetic, is not fully understood. Because of this dramatic effect of folate, the U.S. food supply was fortified for folic acid in 1998. Concerns have been raised about potential adverse effects of "excess" folic acid intake in the U.S. population as well as other concerns about the level of fortification being insufficient to maximize the reduction in birth defects. Current work on the genetic basis for the folate effect on NTDs will be a primary effort of our investigators and is expected to result in the identification of "at-risk" individuals.

One concern expressed in relation to high folic acid intake is the masking of anemia in the elderly with the consequent development of neurological disease in B12-deficient individuals. Impaired status of various vitamins including B vitamins and antioxidants as well as antioxidant bioactive food components have been implicated in impaired cognitive function. Our efforts to elucidate the optimal intake and/or exposure levels of select dietary constituents (vitamins and phytochemicals) will play a critical role in advancing knowledge resulting in the maintenance of optimal cognitive function in the elderly.

Importance of Work: There is significant need to further understand the role of nutrients and phytochemicals and their interactions in reducing the risk of chronic disease. This work must encompass a clear understanding of the factors that determine the bioavailability and targets of action to be able to make recommendations to specific stakeholders and ultimately affect the health of our nation. The opportunity for our experts to share knowledge, techniques and resources is central to advancing our understanding in a timely, resource-efficient and strategic manner. For example, while researchers in bone health focus on vitamin D as a nutrient of relevance to bone formation, others in cancer research are able to appreciate the role of vitamin D and to rapidly advance our understanding of its functional potential in cancer prevention through collaborations only possible through this project. Other examples of this rich cross-fertilization is the dual preventive potential of folate in cognitive disease and cancer risk reduction, or soy isoflavones as a modifier of disease risk in cancer, metabolic disease and bone-related disease.

Technical Feasibility of Studying Bioavailability and Bioactivity of Food Components: This W2002 multistate group is an extremely diverse group with unique expertise that is ideally poised to identify the mechanisms and bioavailability of nutrients and other bioactive food components on chronic disease prevention. All of the proposed projects involve ongoing methodologies in the respective investigators' laboratories and through this collaborative effort, are extremely feasible (see related and previous work). W2002 researchers have established an international reputation in studying the mechanisms and bioavailability of nutrients and other phytochemical components important in chronic disease prevention. We have extensive experience in kinetic modeling of nutrients, bioavailability studies in rodents and humans, epigenetic and genetic polymorphism studies, as well as significant expertise in the study of obesity, osteoporosis, cancer and inflammatory disorders in both model systems and in the human population. Many of the proposed methodologies were pioneered by W2002 members and have been recognized.

Advantages of a Multi-state Effort for the Study of Bioavailability and Bioactivity of Food Components: Through this multi-state effort we are able to examine mechanisms at the molecular cellular level and directly translate these effects to the population at large. This type of effort and scope would not be possible without the collaborative expertise of each multistate station. Defining the bioavailability and function of bioactive dietary chemicals in chronic disease is complex and requires a multifactorial approach. No single station has the capacity, resources, or equipment to undertake the proposed work in isolation. Through this multistate effort, we are able to combine efforts and share resources that are unique to each individual station. The issues to be addressed and benefits to be achieved are common to the nation as a whole, not a single state. Since several of the issues are being addressed in different ways among researchers in several states, information exchange and collaborations will facilitate goal achievement, limit duplication of effort among members' respective units, and potentially produce new and innovative methodologies for understanding the metabolism and roles for these bioactive food compounds in human health. The diversity of expertise (from basic science to human population studies to outreach/extension) among faculty at major land-grant universities facilitates translational studies. Data arising from W2002 collaborative activities will be disseminated to the greatest extent possible among stakeholders and will thus provide maximum benefits to the U.S. public. W2002 efforts and focus are not duplicated in any other regional project.

Impact of Research/Endpoints: We expect the multidisciplinary, translational research supported by the W2002 investigators to have significant impact on health, which will be identifiable as measurable endpoints. In a general sense, we expect select aspects of this research to result in the identification of new nutrition based prevention strategies for cancer, obesity, osteoporosis and other significant chronic diseases. More specifically, we expect this work will help establish more effective biomarkers for nutrient status and new dietary requirements in the population. For example, W2002 researchers have been conducting research in folate and/or vitamin D metabolism and requirements which are expected to re-define requirements (DRIs) for these nutrients in the context of chronic disease risk reduction in the future. Further, collaborations in the area of cancer are expected to result in the development and testing of new chemoprevention agents and dietary recommendations that will impact oncology patient care in the future. If this work is not completed, this would markedly hamper our understanding of the metabolism and mechanistic role of each nutrient in maintaining optimal health. Without the research proposed by this multistate group, human nutrient requirements cannot be assessed, which might lead to inadequate approaches to the prevention of chronic diseases such as cancer, obesity and osteoporosis. Other endpoints for the W2002 group include more tangible items such as collaborative research grants, publications as well as outreach endpoints such as support of local extension staff and faculty in developing important patient/public-oriented educational materials integrating our research findings.


Because of the interactions and collaborations of W2002 scientists, agriculture and health related entities have been provided with considerable information for use in the prevention of nutrition-related chronic diseases, consistent with regional and federal priorities. This group has published over 290 articles in the last 5 years. Data related to previous priorities addressed in W2002 research has served, in part, as the basis for:

* the fortification of cereal grains with folate to decrease the risk of neural tube defects;

* the inclusion of health statements about calcium and osteoporosis, and hyperhomocystinemia and heart disease on food labels; and

* the increased DRIs for calcium for young adults.


This research is innovative and novel in that it utilizes a translational approach to understand the role of nutrients, especially those consumed as a "whole food" on optimal health. The collaboration among basic, clinical, epidemiological and extension faculty from diverse academic institutions across the U.S. affords the opportunity to advance scientific understanding of the role of, and requirements for, nutrients and bioactive food components in maintaining optimal health. Further, our robust scientific interactions, which are possible through this collaborative agreement, afford an opportunity to translate research findings to public health practice in a time and cost-efficient manner.


CRIS Search: A CRIS search with key words bioavailability & nutrients and phytochemical or phytonutrient revealed 18 active projects. The committee is familiar with all projects. There are no similar regional projects.


A recent review of active multi-state projects suggests only one project has the potential for overlap with our efforts, W2122, "Beneficial and adverse effects of natural, bioactive dietary chemicals on human health and food safety." However, there are several important distinctions between the projects. First, this project focuses on cancer, obesity, osteoporosis, neural tube defects and cognitive function while W2122 addresses issues related to cancer, foodborne toxins, immunity and antimicrobials. Food safety is the central theme of their research and is not included in our focus in any definitive way. In fact, the one area of potential overlap - cancer - is approached very differently in the two applications. Our proposed project continuation uses a collaborative translational model to identify and implement effective dietary strategies involving select nutrients and phytochemicals to reduce risk while the W2112 investigative team employs basic science techniques alone to more clearly characterize the role of select bioactive food compounds (BAFC) in promoting cancer risk or modulating risk associated with exposure to food-based toxins such as aflatoxin or heterocyclic amines. Another potential overlap is in the area of neural tube defects (NTD); however, here we are addressing the role of B vitamins in prevention and the W2112 group is identifying potential neurotoxins in crop feeds that contribute to risk. Further, a search of related research within the USDA CRIS database suggests there are individual investigators completing related research, including work on BAFC in cruciferae (UCBerkeley), chlorophyll (UHawaii), bitter melon (UHawaii), soy (UIllinois), bioidentification methods for BAFC (UCDavis), and several projects related to quantification of BAFC in select crops/foods (Beltsville). The vast majority are basic and food science related with few indicating the translational approach to phytochemicals research proposed here. Moreover, issues regarding bioavailability and potential interactions among food, nutrients and processing are not addressed by the W2112 group.


Without question, the information derived from the proposed research is innovative and unique, and the proposed dissemination of information to both the scientific community and lay public will provide part of the framework on which future nutrient recommendations can be based. Table 1 outlines a list of proposed nutrients and phytochemicals for initial analysis, their biomarkers and endpoints/clinical outcomes.

Related, Current and Previous Work

W2002 researchers have worked collaboratively and have made significant contributions to understanding the role of nutrients and bioactive food components in optimal health. To continue in these efforts our research team will focus efforts in reducing the risk of chronic disease and developmental/degenerative disorders through the examination of nutrient/phytonutrient bioavailability, their mechanisms of action and the extent to which genetic and epigenetic alterations effect individual nutrient/phytochemical needs and chronic disease risk.


Bioavailability of nutrients and bioactive food components.

A. Absorption and Metabolism

Calcium. Dairy products supply 72% of the calcium in the adult US diet, grain products about 11%, and vegetables and fruits about 6% (USDA, DHHS-89-1255). Although beans and corn tortillas processed with lime provide the bulk of dietary calcium for some ethnic groups, it is difficult for most to ingest sufficient calcium from foods in a cereal-based diet without liberal consumption of dairy products. Many have turned to dietary supplements to meet their calcium needs. In response, food manufacturers have developed calcium-fortified products (e.g., orange juice, rice, breads, low or non caloric beverages) that have had a range of market success. IN have developed a nonlinear regression model using data in adolescent girls to study the relationship between calcium intake and calcium retention (Jackman et al., 1997). Recently, IN has studied this relationship in adolescent boys and racial differences (Braun et al, a&b). The effect of other dietary constituents and exercise on this relationship and predictors of variance in calcium retention needs to be addressed and is a focus of work done in IN.

Folate. Folate functions in transporting single carbon fragments from one metabolic intermediate to another in the synthesis of nucleic acids and in the metabolism of amino acids. Folate deficiency leads to impaired cell division that presents as neural tube defects and anemia and to dysfunctional amino acid metabolism that presents as hyperhomocystinemia that is now widely accepted as a major risk factor for heart disease. So folate nutrition status is constantly monitored, but measuring erythrocyte folate has been a vexing problem for a very long time until the CA-D station developed an accurate, precise, reliable, and high throughput method, 5000 samples in 21 days (Owens JE et al., JAFC 2007;55:3292-7). Erythrocyte folate is the best indicator of folate status because it reflects the intracellular pool. The NHANES has 5000 samples analyzed each year to monitor changes in folate status over time, now this can be completed in three weeks. The CA-D station also developed and validated a folate specific folate screener to assess folate intake that can be completed in 10 minutes (Owens JE et al., JAFC 2007; 55:3737-40).

Current knowledge of folate bioavailability is based largely on research conducted by W-2002 scientists. Current estimates of the efficiency of dietary folate absorption are variable depending on the experimental protocols used to measure absorption (Rodriguez et al., 1978). Now a better understanding of factors that affect the bioavailability and dynamics of folate metabolism among individuals is needed for future revisions of the DRI/AI for folate. Current work in CA-Berkeley and CA-Davis have developed novel technologies and modeling systems to address these issues with folate and other B vitamins.

Folate and its role in methylation have also been a topic of active research for cancer research. Growing epidemiological evidence suggests that requirements for folate may be "u-shaped" in terms of protection against cancer, suggesting that intake at either extreme could lead to cancer promotion. Existing datasets will provide further insight into this complex pattern of optimal nutrient exposure.

Vitamin E. Greater than 90% of Americans fail to meet the estimated average requirements for dietary vitamin E (Maras et al., 2004). Therefore, continued studies at CT and UC-D are aimed at assessing vitamin E absorption, distribution, and metabolism. CT has utilized LC/MS techniques for assessing stable isotopic vitamin E administration in humans to evaluate vitamin E absorption, antioxidant recycling, and oxidative stress-mediated vitamin E pharmacokinetics. UC-D has constructed a kinetic model of human vitamin E distribution, fluxes, pool size distribution and P450-mediated metabolism as it might occur in vivo. These stations will initiate collaborations in this area to extend their observations and ascertain future dietary recommendations.

Green tea catechins. In progress studies at CT suggest that green tea extract, presumably through the bioactivities of polyphenolic catechins, may regulate intestinal lipid absorption, lipid metabolism and may serve as a dietary strategy to attenuate the development of nonalcoholic fatty liver disease (NAFLD). Continued studies in this area are aimed at evaluating the bioavailability and metabolism of green tea catechins in experimental animal models of obesity. Continued studies in this area in CT are aimed at evaluating the bioavailability, metabolism, and mechanisms of action of green tea catechins in experimental animal models of obesity and defining the mechanisms underlying the inhibitory effect of green tea extracts on lipid absorption. Preliminary work at AZ indicates that overweight, post-menopausal women provided with decaffeinated green tea had a significant, short-term (30 minute) increase in resting energy expenditure that while of importance is too short in duration to suggest any long term role in weight control. Collaborative intellectual interactions among investigators working with tea research should promote greater understanding of the potential use of tea and tea polyphenols in obesity prevention.

Isothiocyanate and Indoles. Isothiocyanates and indoles derived from cruciferous vegetables such as sulforaphane and indole-3-carbinol have been studied extensively for their potential health benefits including cancer prevention, anti-inflammatory activity and immune dysfunction (Ho et al., 2011). However, the bioavailability of these compounds and their metabolites in humans, especially to target tissues, is relatively unknown. Researchers in OR have developed sensitive mass spectrometry methods to better understand the distribution of sulforaphane and indole-3-carbinol and their respective metabolites using pre-clinical models and human subjects. Researchers have also studied effects of whole foods versus supplement sources and have recently found that supplements that lack myrosinase, a key enzyme that helps release sulforaphane, markedly decreases bioavailability of the compounds. Several cancer clinical trials are also underway to test the effects of sulforaphane supplementation. Factors such as polymorphisms in metabolizing enzymes such as glutathione-S-transferases and the gut microbiota may also alter bioavailability and determine individual responses to these phytochemicals (Clarke et al., 2011). Research in this area is important for determining optimal dietary recommendations for disease prevention.

Biotin. The Food and Nutrition Board acknowledges that human biotin requirements are unknown (National Research Council, 1998). Consequently, only recommendations for Adequate Intake are available for biotin. Recommendations for Adequate Intake are based solely on biotin intake in the general, apparently healthy, population (National Research Council, 1998). This approach is flawed in the case of biotin, where dietary intake data are only crude estimates. Currently, no studies are available that quantified biotin in foods by using chemically specific assays (Zempleni and Mock, 2000), and it is unclear whether intake estimates exceed or underestimate the true biotin intake. NE made important contributions to quantifying human biotin requirements by showing that biotin is 100% bioavailable (Zempleni et al., 1999a) using chemically specific assays based on protocols developed in NE (Zempleni et al., 1996; Zempleni and Mock, 1999b). NE has submitted a manuscript that critically assesses the usefulness of markers for biotin status in humans, based on a cost-efficient outpatient feeding protocol developed in NE (Eng et al., submitted). This paper provides evidence that the abundance of biotinylated carboxylases in blood cells is the only marker that reliably distinguishes biotin-deficient, biotin-sufficient, and biotin-supplemented individuals.

B. Nutrient/Gene interactions affecting nutrient requirements.

Inefficiencies in folate metabolism and inadequate intake lead to poor folate nutriture with increased risk of degenerative and developmental diseases. The determination of folate needs in humans has been problematic and in particular, there is a need to understand the effects of common single nucleotide polymorphisms (SNP) on B-vitamin metabolism (Clifford et al., 2004, Lin et al., 2004). Previously at CA-Davis, they constructed a kinetic model of in vivo human folate metabolism in 13 adults administered a 0.5 nmol oral dose of 14C-folic acid. The model estimated that apparent absorption of folate was 80%, enterohepatic recycling was 5350 nmol/d, and total body folate stores were 225 mol. All the estimates were larger than previous ones. So, in a preliminary experiment (study 1), we tested whether single nucleotide polymorphisms (SNPs) in folate-relevant enzymes affect folate metabolism by genotyping the 13 subjects SNPs in the following enzymes: MTHFR C677T and A1298C, MTR A2756G, MTRR A66G, CBS T833C, GCP II C1561T, TC II C776G, and RFC1 A80G and testing their effects on folate metabolism. Results of study 1 suggested that SNPs in MTHFR C677T, MTRR A66G, TC II C776G, and RFC1 A80G had significant effects on folate metabolism as reflected by RBC folate, plasma homocysteine and serum B12. To confirm our results, 370 additional subjects were genotyped for SNPs in the same enzymes out of which 234 Caucasian subjects were used to evaluate main and two-way interaction effects of age, gender, BMI, and genotype on plasma normalized homocysteine (nHcy) and on serum vitamin B12. Four main and two interaction effects affected nHcy, whereas two main and one interaction effects affected serum B12. Kinetic and statistical modeling of metabolic and genetic data can refine folate needs for optimal health and potential interactions with genotype. Research by W2002 scientists has created cutting edge methodologies to understand the impact of polymorphism on folate metabolism to help understand individual folate needs.

NE took an alternative approach to characterizing effects of SNPs on nutrient metabolism. The enzyme holocarboxylase synthetase (HLCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Low activity of HLCS causes de-repression of long-terminal repeats, thereby impairing genome stability and increasing cancer risk (Chew et al., 2008). NE relied on biochemical approaches as opposed to using population-based approaches for investigating the effects of SNPs in HLCS on the catalytic activity (Esaki et al., 2012). Five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS were selected for these studies. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96L SNP did not alter enzyme kinetics. These findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.

The ability to study complex food/metabolic interactions that determine nutrient/phytochemical availability is an additional strength of this W2002. Previous work in OH and IN have established the role of salivary enzymes and gut microbes on phytochemical metabolism and bioavailability. Studies in OR and CO have also established differences in bioavailability from whole food sources compared to individual supplements (Hsu et al., 2010 and Clarke et al., 2011), and have determined that food processing also alters phytochemical bioavailability, respectively.

Nutrients and other food components-Mechanisms of bioactivity

A. Bioactive food components and maintenance of optimal health

Cancer prevention (cruciferous foods, biotin, tea, soy)

The role of diet and bioactive food components to modify cancer risk is an area of active research yet improvements in dietary measurement, increased evaluation of dietary interventions to reduce risk for cancer and more frequent use of surrogate biomarkers of disease risk in the context of dietary intervention research are needed. Foods rich in bioactive food components, including carotenoid-rich yellow-orange vegetables and fruit, cruciferous vegetables and citrus have been hypothesized to reduce cancer risk through a variety of bioactivities including reduction in oxidative stress. Currently in OR, two clinical trials in both breast and prostate cancer patients are ongoing using broccoli extracts.

Green tea has been studied for its chemopreventive properties for well over a decade including its role in modulating oxidative stress, inflammation, apoptosis, etc. Researchers in this group are involved in a number of studies testing the bioactivity of green tea. AZ has recently completed a short-term green tea intervention trial among overweight breast cancer survivors to assess the effect of intake of 4 cups green tea daily for 6 months, as compared to placebo tea, to test if it will reduce body weight, body fat, insulin resistance and inflammation. OR have also found a decrease in inflammatory signal pathways with green tea consumption in a hormone-induced model for prostate cancer (Hsu et al., 2010 and Wong et al., 2010).

Epigenetics is the study of heritable features not associated with changes in the nucleotide sequence of DNA. These features are potentially reversible and may affect genomic stability and expression of genes, including tumor suppressor genes and oncogenes. Hence, epigenetic mechanisms have crucial roles in cancer risk. NE has demonstrated that HLCS enters the nuclear compartment, where it binds to chromatin and catalyzes the covalent attachment of biotin to histones H3 and H4 (Camporeale et al., 2004, Kobza et al., 2005, Kobza et al., 2008, Singh et al., 2011, Bao et al., 2011). Low activities of HLCS and biotin depletion cause severe phenotypes such as short life span and low heat resistance in Drosophila melanogaster (Camporeale et al., 2006) and de-repression of long terminal repeats and genome instability (Chew et al., 2008). Biotinylation of histones is a rare event, rendering it difficult to attribute severe phenotypes to low levels of histone biotinylation (Stanley et al., 2001, Kuroishi et al., 2011). NE has developed a model suggesting that the effects of HLCS deficiency and biotin depletion in gene regulation are caused by physical interactions between HLCS and other chromatin proteins such as methylated DNA-binding proteins, histone methyl transferases, and histone deacetylases (Kuroishi et al., 2011, Xue and Zempleni, 2011, Li et al., submitted). These observations provide plausible links between biotin metabolism and cancer risk.

Research in OR suggests that targeting histone modifications or DNA methylation by dietary bioactives is a novel mechanism for prostate cancer prevention (Myzak et al., 2005, Myzak et al., 2006, Dashwood et al., 2006). In particular, OR has found that sulforaphane and indole-3-carbinol, an isothiocyanate derived from cruciferous vegetables is also an HDAC inhibitor that slows prostate cancer growth in the cell, animals and humans (Clarke et al., 2011 and Beaver et al., 2012). The identification of dietary HDAC inhibitors, and their use either alone or in combination, may increase efficacy of anti-cancer therapies/prevention strategies.

Bone Health (Calcium, iron, vitamin D, isoflavones, prebiotics)

Several types of botanical products containing isoflavones are being marketed as an alternative to estrogen replacement therapy to prevent osteoporosis in postmenopausal women. Estrogen suppresses bone resorption. Efficacy of isoflavones on bone health has mixed results which may depend on the dose and interaction of isoflavones and the ability of individuals to produce bioactive metabolites through gut microflora. IN will use 41Ca, a long-lived isotope, to label the skeleton and an Accelerator Mass Spectrometry (AMS) to monitor perturbations by diet. This innovative approach to study bone resorption is a direct measure of bone resorption in contrast to biochemical markers of bone turnover and traditional calcium kinetics, compared to studying changes in bone density, and is not as invasive as bone histomorphometry. The effect of various types and dosages of isoflavone-containing botanical products sequentially will be tested in the same postmenopausal women for their effectiveness in suppressing bone resorption by measuring changes in 41Ca excretion in the urine. IN published the first clinical trial using this method, a dose response study of soy isoflavones in postmenopausal women (Cheong et al., 2007). A dose response effect of genistein, kudzu, and equol on bone and bone quality in an ovariectomized rat model and in postmenopausal women and the influence of gut microflora will be determined in IN. IN and UCD have developed the ability to prepare C-14 and C-13 labeled plant tissue culture. Pharmacokinetics and the metabolic fate of bioactive compounds throughout the body can be measured and kinetic and compartmental models developed. OK has also developed advanced microCT methodologies to examine bone architecture and bone density alterations with various dietary approaches.


Obesity (tea, vitamin E, biotin)

The role of green tea in reducing obesity and the detrimental health affects thereof has been an additional interest of these investigators. CT and OR are actively investigating the role of green tea extract in regulating the inflammatory and oxidative stress triggered processes in the development of non-alcoholic fatty liver disease (NAFLD). Preliminary results suggest that green tea extract attenuates hepatic steatosis and hepatic injury in a mouse model of obesity-triggered NAFLD. Continued studies are underway to unravel the protective mechanisms by which green tea extracts (1-2% green tea extract powder containing 30% green tea catechins) attenuated hepatic steatosis and injury in obese mice. Additional studies are in progress to evaluate specific vitamin E forms (a- vs. g-tocopherol) in attenuating the oxidative stress and inflammatory processes implicated in the pathogenesis of NAFLD.

NE discovered anthocyanins in grape juice, grapes, and grape leaf extracts that inhibit the differentiation of mesenchymal stem cells (bone marrow cells) into adipocytes by ~80%. These effects are caused by a loss in acetyl-CoA carboxylase 1 and/or 2 activities, due to a loss of HLCS-dependent biotinylation of these carboxylases. The effects can be reproduced in whole organisms such as Drosophila melanogaster. When a Drosophila mutant (brummer mutant) that is prone to accumulation of large amounts of body fat, is fed a diet containing grape leaf extracts, total body fat decreases by ~40% in male flies and ~25-30% in female flies compared to controls fed a grape-free diet. NE has initiated discussions with CO to expand this line of research to include potatoes.

Objectives

1. Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and other food components.
   
2. Evaluate the bioactivity of nutrients and other food components in order to elucidate their underlying protective mechanisms.
   

Methods

Objective 1: Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and other food components. 1. Absorption and Metabolism W-2002 scientists have developed state of the art techniques for assessing all components of bioavailability for several nutrients and for bone turnover. Novel methodologies include use of rare and stable isotopes for calcium, and 14C labeled bioactives (IN) and vitamins such as folate (UCD) and vitamin E (CT). The use of these isotopes allow for W2002 scientists to precisely monitor each nutrient, its uptake, distribution into plasma and elimination in urine & feces across different lifestage and physiological conditions. For example, work done in IN has effectively modeled calcium intake and its metabolisms during adolescence. This data will be critical in helping establish DRI for calcium in this age group. W2002 scientists can also examine alterations in nutrient metabolism during different physiological conditions. Research done in CT has found altered metabolism of vitamin E with smokers. Researchers in UCB together with UCD investigated the influence of folate and vitamin B12 status in our experimental animals using cDNA array technology and have identified a number of inflammatory response genes that are responsive to vitamin status. W2002 scientists will continue development of these methods and use them to study bioavailability and factors affecting vitamin, mineral, and bioactive compound distribution and metabolism at IN, UCD, UCB, CO, OH, and OK. An exciting research area is exploring the interactions between the gut microbiota and nutrient bioavailabity and metabolism. 2. Modeling and model development Kinetic modeling and compartmental analysis will be used to assess vitamin, mineral and bioactive compound turnover rates, pool sizes, and metabolism (IN, UCD). Bone turnover rates will be used to evaluate predictors of bone formation and resorption during growth and age-related bone loss (IN). Modeling will be used to predict outcomes and design experiments to test predicted outcomes. For example, we will determine the bioavailability (absorption, distribution, metabolism, elimination) of 5-MTHF by quantifying the dynamic and kinetic behavior of tracer doses of 14C-Folic acid and 14C-5-methyltetrahydrofolate (14C-5-MTHF) using a test/re-test design in adults (UCB, UCD). Using AMS and compartmental modeling, differences in the dynamic and kinetic behavior of each folate form as it might occur in vivo in humans will be determined. This approach to bioavailability (absorption, distribution, metabolism, elimination) is unique because we will quantify the kinetic behavior of 14C-folates under steady-state conditions for long time periods to ensure that the administered tracers are fully equilibrated with the most slowly turning over pools; these pools determine the kinetic behavior in plasma. This can only be done by utilizing AMS to analyze the amount of 14C in plasma, urine, feces, and RBCs. Key parameters that we will determine include whole body turnover, mass balance, half-lives for absorption, distribution and elimination in plasma, and the cell population kinetics of RBCs followed over their entire life-span of 120 days. Because we have already constructed a kinetic model of FA metabolism, we are positioned to quantitatively compare the metabolism of the major naturally occurring food folate 5-MTHF to FA. Direct comparisons of 5-MTHF to FA will aid in the development of dietary recommendations for optimal health. 3. Genetics Gene polymorphisms that affect nutrient flux through metabolic pathways may well be important determinants of nutrient requirements for optimal health. Genetic polymorphisms that are associated with traits such as hyperhomocysteinemia, hypercholesteremia, etc., may be well normalized (over-ridden) by appropriate dietary adjustments. Initially, folate and vitamin B12 will be further studied (UCD and UCB). The influence of polymorphisms in glutathione-s-tranferases and cytochrome P450, on bioavailability of phytochemicals found in cruciferous vegetables will also be examined in collaboration between AZ and OR. Modeling has already suggested that genetic polymorphisms involved in folate metabolism affect the flux through several pathways and these model changes will be tested in experimental systems (UCB and UCD). Tissue specific and cell compartmental models are being used to predict effects of polymorphisms in folate genes on metabolic fluxes (UCB). We will study subjects for the folate relevant SNPs and search for additional folate-relevant SNPs of RFC1 using a SNP discovery platform developed by re-sequencing candidate genes in panels of 24 subjects from a population of human subjects screened for eight SNPs, diet, and blood chemistries. 4. Development of facile methods for assessing nutrient status and bioavailability Equations for simple methods to predict true absorption of nutrients such as Ca (IN) against full kinetic models are being developed. Genetic polymorphisms are being screened as predictors of folate status (UCD and UCB). At the end of the project we will have kinetic models of the interactions of SNPs on 5-MTHF versus FA metabolism in humans that will clarify inter-individual responses for the development of improved strategies to minimize the risk of NTDs, age-related diseases and poor folate status. We may also have newly identified SNPs of the RFC1 that affect folate homeostasis. In summary we will know whether the main and interactive effects of genetic polymorphisms in folate relevant enzymes are significant determinants of the dynamic and kinetic behavior of folate (natural versus synthetic folate) metabolism as it might occur in vivo in humans. We will also have models for absorption of other minerals, such as calcium, that will be used to help define absorption of these nutrients in humans and help develop DRIs for optimal health. Importantly, all of the goals proposed in Objective 2 are translational studies performed in human subjects, which is a major strength of this project. Objective 2. Evaluate the bioactivity of nutrients and other food components in order to elucidate their underlying protective mechanisms. 1. Identify the relationships among nutrient status, dietary intake of nutrients and other food compounds and their association with chronic disease risk in the human population. Collaborating investigators will utilize existing and yet to be developed data sets from cohort studies and clinical trials to assess the relationship between nutrients/ bioactive food components and disease outcomes and/or surrogate biomarkers of disease risk. Clinical and biological samples from the clinical trials using broccoli sprout extracts in OR to evaluate the role of cruciferous vegetables and their constitutive bioactive food components (OR) in reducing breast cancer recurrence and prostate cancer risk will be utilized. In addition, dietary and clinical data from over 165,000 post-menopausal women from the Women's Health Initiative, that include sub-groups randomized to a low fat diet, and/or calcium supplementation, will be used to assess relationship between nutrient status and health outcomes. In collaboration with other investigators with expertise in select nutrient metabolism, including iron (AR), calcium (IN), folate (CA-B), vitamin E and tea polyphenols (CT), this longitudinal dataset which includes cancer, cardiovascular, and bone health outcomes, supports our collaborative efforts to assess the role of diet in chronic disease risk reduction through epidemiological data analysis. To advance our capacity to evaluate the relationship between bioactive food components and disease risk, we will also develop and enhance dietary assessment tools of intake and/or exposure to food components. This includes development of nutrient-specific self-reporting instruments (AR) and assessment of biological markers of intake including: carotenoids (AR), calcium (IN), vitamin E and polyphenolic flavonoids (CT), folate (CA-D, CA-D). 2. Identify interactions among dietary compounds and genetic and epigenetic events that influence growth, developmental disorders, and chronic disease. The effect of genetic variation on bioactivity of bioactive food components will be assessed. Initially, folic acid, vitamin B12, biotin, and glutathione S-transferase (GST) polymorphisms will be studied (CAD, CAB, NE, and OR). In addition, a collaboration among NE, OR and AZ will enable the study of epigenetics and cancer risk. NE will examine the effects of biotinylation of histones on cancer risk. Biotinylation will be assessed using chromatin immunoprecipitation and real-time PCR and chromosome painting techniques will be used to quantify effects of altered histone biotinylation in pericentromeric chromatin on chromosomal stability. In addition, a mouse model for disrupted histone methylation has been developed at UC-B and will be used to study nutritional modification of methylation (in development). The impact of cruciferous vegetable intake and other bioactive food components on histone acetylation has not been clearly identified. Importantly, collaboration between OR and AR give the opportunity for TRANSLATIONAL studies, using a unique and extensive longitudinal dietary, clinical and demographic dataset, tumor tissue samples, and DNA samples collected for the on-going Women's Healthy Eating and Living (WHEL) Study of 3088 breast cancer survivors participating in a randomized, controlled, plant-based dietary intervention trial. With this group, we will be the first, to our knowledge, to refine our understanding of epigenetic events by describing the interaction between epigenetic alterations (HDAC) and candidate genetic polymorphisms in relevant genes involved in the bioavailability, bioactivation and biotransport of cruciferous vegetable-derived isothiocyanates including sulforaphane, cruciferous vegetable intake and breast cancer recurrence. 3. Identify bioactive food compounds that regulate antioxidant status, inflammation and cell signaling pathways leading to disease prevention. Specific goals for this aim include, defining the bioactivity of green tea extract on lipid metabolism during the development of obesity-triggered NAFLD and defining the redox sensitive mechanisms regulated by bioactive food components such as green tea, soy isoflavones and fruit and vegetable intake on the development of NAFLD (CT) and prostate cancer (OR). This hypothesis will be tested by feeding green tea extract to ob/ob mice (model for NAFLD) or hormone-induced prostate cancer. CT and OR work in collaboration to examine dietary antioxidant status, endogenous antioxidant defenses, inflammatory cytokines and oxidative/nitrative stress, damage biomarkers, redox sensitive transcription factor analysis and gene expression of factors regulating inflammation and cell signaling. Work done on other natural phytochemicals, such as those found in blueberries is being investigated in ME. We will also assess the effect of nutrients and/or food components on oxidative stress and inflammatory biomarkers in a human population. Markers of oxidative stress (8-OHdG and 8-epi-PGF2 alpha) and inflammatory (hs-CRP, TNF) will be measured in pre- and post dietary intervention groups (AR, IN, OR, CT). 4. Evaluate bioactivity in bone health Life stage and health status on skeletal calcium and bone retention. The methodology utilized in rodent studies will be translated to studies performed in human subjects to examine effects of life stages and other nutrient deficiencies on bone health. Adolescents will be studied (IN) for effects of vitamin D, calcium source, and subgroup populations. Postmenopausal women will be studied for effects of plant isoflavones (IN, AR) and gut microflora (IN) on calcium bioavailability and bone loss by DEXA (bone density ) and microCT (bone architecture). Similarly, breast cancer survivors will be studied for plant-based versus meat-based diets (AR), and their influence on bone density. Ethiopian mothers and infants will be studied for zinc and vitamin D status (OK). Biomarkers for zinc status and vitamin D status will be correlated with alterations in bone density and architectural alterations in bone in these populations. Expected endpoints/outcomes: The expected endpoints resulting from the multidisciplinary, collaborative interactions among the W2002 investigators include: *Establish models of bioavailability for key nutrients involved in optimal health, including calcium, vitamin D, B vitamins, Vitamin E and phytochemicals such as isoflavones and isothiocyanates. *expanded use of existing epidemiological datasets for which the collaborative researchers will have access in order to test hypotheses related to the role of our targeted bioactive food constituents in chronic disease risk reduction *develop collaborations to translate in vitro and animal studies on nutrients to human populations *develop collaborations to perform mechanistic studies from observational epidemiological studies *collaborative support in grant development to assure optimal research design particularly in regard to bioactive food constituent measurements, both through self report and biological markers of exposure *Shared methodology regarding measurement of oxidative stress and/or inflammation as an intermediate indicator of chronic disease risk, including strengths and limitations of available biomeasures, understanding of bioactive deposition requirements to modulate these markers and optimal measurement conditions including frequency as well as in relation to timing of exposure *Resource sharing related to new imaging technologies for use in assessing endpoints of feeding and/or bioactive dietary constituent epidemiological trials (i.e. bone health, body composition, nutrient deposition in targeted tissues) *through shared knowledge, advanced understanding by all investigators of the potential role of genetic background in modifying exposure and/or efficacy of bioactive dietary constituents in relation to normal growth as well as chronic disease risk reduction *joint peer-reviewed publications *support for the development of targeted outreach materials that integrate research findings resulting from this research *The W2002 group has developed a website describing the research interests of its members. The website is currently hosted by the Nebraska Gateway to Nutrigenomics (nutrigenomics.unl.edu) until a permanent home has been identified. The website will be used for disseminating research findings (eMaterials) and for outreach activities after re-authorization of this group. Summary A summary of proposed collaborations and interactions are listed in table 2. We have also included specific expertise each station member brings to this collaborative effort in table 3-Resources. This W2002 team of investigators consists of a unique group of researchers that can advance science in defining the role of bioactive dietary constituents for optimal human health. This group includes experts in a wide variety of nutrients and bioactive food constituents - an appropriate approach given that the food we eat is a complex and integrated mixture of nutrients and bioactives. Further, the research technological expertise of our group is also diverse and includes expertise in cell culture, tissue culture, animal models, imaging, epidemiology and clinical trials. This diversity provides fertile ground for collaborations in the form of informal conversations as well as formal and collaborative grant proposal development.

Measurement of Progress and Results

Outputs

• Additional collaborative projects
• Research data
• Peer-reviewed publications
• Presentations and posters at scientific meetings
• E-files and web-based applications
• Output 6 Outreach presentations or publications for non-scientific organizations Output 7 Website development Output 8 Annual meeting Output 9 Students advised and postdocs trained

Outcomes or Projected Impacts

• Increased knowledge of the bioactivity of nutrients and other food components and their underlying protective mechanisms
• Increased knowledge of the bioavailability of nutrients and other food components
• Development and publication of models for determining bioactivity and bioavailability of nutrients and other bioactive food components
• Building a highly skilled workforce in human nutrition

Milestones

(2013): Initiate the collaborative studies described for year 1. Raise seed money for supporting research collaborations among group members. Publish peer-reviewed original research articles, review articles, and book chapters. Discuss strategies for expanding e-materials and web-based data distribution. Update the group's website, including e-materials and web-based distribution. Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public.

(2014): Initiate the collaborative studies described for year 2. Raise funding for supporting research collaborations among group members. Publish peer-reviewed original research articles, review articles, and book chapters. Evaluate collaborative efforts on projects initiated in year 1 that extend to all years. Update e-materials and web content. Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public.

(2015): Initiate the collaborative studies described for years 3-4. Evaluate collaborative efforts for bioavailability of calcium, for bioinformatics of genotyping data, for epigenetic cell culture studies and on-going efforts that extend to all years. Publish peer-reviewed original research articles, review articles, and book chapters. Update e-materials and web content.Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public.

(2016): Report on and assess further collaborative efforts for in vivo models, human dietary supplement intervention trials, dietary measurement/validation, cancer survivors dataset and epidemiological and association studies and on-going efforts that extend to all years. Publish peer-reviewed original research articles, review articles, and book chapters. Evaluate the program for e-materials and web-based distribution and make changes as appropriate. Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public.

(2017): Evaluate the accomplishments of the collaborative efforts for the project. Prepare a report on the web-based applications. Determine goals and prepare a proposal to continue collaborative efforts. Publish peer-reviewed original research articles, review articles, and book chapters. Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public.

Projected Participation

View Appendix E: Participation

Outreach Plan

All members of this multistate group will engage in various outreach activities. The type of activity will depend on each member's expertise and environment. For example, some members will make presentations to professional organizations and the lay public, or will have appearances on local TV and radio stations. Other members will offer research and education opportunities for high school students and teachers. All members will disseminate their research findings through publications in science journals, presentations at science meetings, and through invited seminars. Group members will adhere to the practice of making manuscripts available in the public domain no later than 12 months after publication. Since our last re-approval, we have developed a website illustrating our members areas of expertise (http://nutrigenomics.unl.edu/w-2002-multistate-project). The website is currently maintained by Dr. Janos Zempleni (NE). We will continue to expand the information and services offered through the website, e.g., by posting experimental protocols and publications and, perhaps, by offering webinars.

Organization/Governance

The recommended Standard Governance for multistate research activities includes the election of a Chair, a Chair-elect, and a Secretary. All officers are to be elected for one to two-year terms to provide continuity. Administrative guidance will be provided by an assigned Administrative Advisor and a CSREES Representative.

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Attachments

Land Grant Participating States/Institutions

AZ, CA, CT, FL, HI, IA, IL, KS, MA, ME, MO, MT, NE, NJ, OH, OK, OR, PA

Non Land Grant Participating States/Institutions

California-San Diego