Brief Summary of Minutes of Annual Meeting

This group has been very productive this year as illustrated by the publications and by developing collaborations among project members. Objectives of our project and activities addressing these objectives are reported below: Objective 1) Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and other food components and their environmental and genetic determinants. The University of California-Davis (Clifford) has conducted detailed investigations of bioavailability of beta-carotene in humans. Bioavailability estimates for absorption and metabolism in humans range from 2 to 90%. The retinol activity equivalent of ingested beta-carotene has an equally broad range. The wide ranges highlighted a major gap in our quantitative understanding of the in vivo human beta-carotene metabolism and its retinol activity equivalent. So that gap was filled by quantifying and interpreting the dynamic and kinetic behavior of metabolism of beta-carotene and its metabolites (retinyl ester and retinol) as it occurs in vivo in humans: using 14C-accelerator mass spectrometry (14C-AMS), genotyping, and modeling. A wide range in the inter-individual bioavailability of b-carotene was confirmed. This wide range had a genetic component as evidenced by single nucleotide polymorphism (SNPs) in lipoprotein lipase (S447S), beta-carotene mono-oxygenase I and/or mono-oxygenase II. Development and optimizing of high throughput 14C-AMS (the ultimate tool for 14C tracer studies in vivo in humans) for biological, biomedical, and environmental applications was key to the success of the project and is rapidly becoming of considerable interest for microdosing of nutrients, phytonutrients, and the development of new drugs and pharmaceuticals. The University of California-Berkley (Shane) has continued studies on the metabolic and nutritional effects of common polymorphisms in human folate-related genes that have been shown to influence disease risk. B12-dependent methionine synthase (MS) and methylene-tetrahydrofolate reductase (MTHFR) genetic mouse models have been evaluated to mimic the effects of these polymorphisms and to evaluate their effects on metabolism and how this is modified by nutritional status. A mouse model has been developed that mimics the clinical effects of human B12 and folate deficiency, and which will allow investigation of potential adverse effects of high folate intake. Evaluation of genetic risk factors for neural tube defects and identification of putative modifier genes which influence folate status, homocysteine levels, and methylation potential continues. 2. Evaluate the bioactivity of nutrients and other food components in order to elucidate their underlying protective mechanisms. The University of Arizona (Thomson) has tested efficacy of vegetables to reduce oxidant stress and inflammation in at-risk overweight women. A significant dose-specific rise in plasma carotenoids in response to escalating dose/intake of the study provided vegetables (doses of 2, 5 and 10 servings daily), suggested excellent dietary adherence and was consistent with self-reported measures of adherence. Results of biomarker analyses suggested that change scores pre-post each feeding dose were not significantly different overall. However, the post-treatment high sensitivity C-reactive protein (hsCRP) was significantly lower, controlling for plasma carotenoid*dose intercept, in women presenting with BMI below 30 kg.m2 (p=0.01). No significant associations were shown for 8-epiprostaglandinF2±. Changes in blood pressure, particularly systolic measures, were also found. Future analyses will include additional inflammatory and oxidative stress indicators in collaboration with Dr. Bruno at UConn as well as collaboration with Dr. Hord to evaluate measures of vegetable and human nitrate levels in association with change in BP. Other trials with whole foods relevant to this multistate project include on-going trials with high anthocyanin carrots, Mediterranean diet with walnuts and lactation and a Ruby Red Oregon State University (Ho) has focused on the examination of the interaction of bioactive nutrients with both genetic and epigenetic mechanisms related to cancer and chronic disease development. The classic view of cancer etiology is that genetic alterations damage DNA structure and induce mutations resulting in non-functional proteins that lead to disease progression. More recently, the role of EPIGENETIC alterations during cancer has gained increasing attention. In both animal models and humans, dietary zinc deficiency increased DNA damage in peripheral blood cells. Importantly, these functional changes preceded any changes in plasma zinc levels. In animals zinc deficiency caused a selective loss of zinc in the dorsolateral lobe of the prostate; the area that is prone to developing cancer. Together these data suggest that maintaining adequate zinc status could be an important factor for maintain DNA integrity and preventing prostate cancer. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables such as broccoli, is a histone deacetylase (HDAC) inhibitor. It increases acetylated histone levels and has anti-cancer properties in the prostate. A mechanism of chemoprotection by SFN in human colon cancer cells and prostate epithelial cells was identified as inhibition of histone deacetylase (HDAC). SFN specifically targeted HDAC3 and HDAC6 in prostate cancer cells not normal cells . Administration of SFN also selectively induced cell death in cancer cells, but not normal prostate epithelial cells. This work suggests that this phytochemical may have the ability to alter epigenetic events that lead to disease prevention. Michigan State University (Hord) has quantified nitrate and nitrite concentrations by HPLC in a convenience sample of foods. Nitrites are produced endogenously through the oxidation of nitric oxide and through a reduction of nitrate by commensal bacteria in the mouth and gastrointestinal tract. As such, the dietary provision of nitrates and nitrites from vegetables and fruit may contribute to the blood pressure-lowering effects of the Dietary Approaches to Stop Hypertension (DASH) diet. Incorporating analyzed values into 2 hypothetical dietary patterns that emphasize high-nitrate or low-nitrate vegetable and fruit choices based on the DASH diet, yielded nitrate concentrations that varied from 174 to 1222 mg. Exposure concentrations of nitrate and nitrite from breast and formula milk as well as vegetables and fruits can approach or exceed the World Health Organization acceptable daily intake (ADIs) recommendation of 3.7 mg/kg body wt for nitrate. Regulatory limits for consumption of nitrates and nitrites are set due to concern over gastrointestinal cancer risk from processed meats and methemoglobinemia in infants from contaminated well water. Despite demonstrated physiologic roles for nitrate and nitrite in vascular and immune function, consideration of food sources of nitrates and nitrites from plant foods as healthful dietary components has received little attention. Current research projects have estimated potential human exposure concentrations across the life course and seek to understand the role of nitrite in the etiology of colorectal carcinogenesis in animal models. University of Nebraska at Lincoln (Zempleni) is investigating roles of holocarboxylase synthetase (HCS) in histone biotinylation. Biotinylation of histones is mediated by HCS and is an important mechanism to repress retrotransposons. De-repression of retrotransposons impairs genome stability and increases cancer risk. Both biotin and HCS deficiency decrease the enrichment of biotinylated histones at retrotransposons, leading to increased transcription of transposons, increased frequency of retrotranspositions, and increased incidence of chromosomal abnormalities. HCS is a chromatin protein but lacks a DNA-binding motif. HCS was found to interact physically with histone H3 to mediate chromatin binding of HCS. Binding of HCS to histone H3 caused biotinylation of lysine residues K9 and K18 in H3. Finally, a HCS knockdown Drosophila melanogaster was generated and showed that HCS deficiency decreases life span and heat stress resistance. Collectively, these studies provided evidence for the biological importance of histone biotinylation and generated insights into the mechanisms mediating the binding of HCS to chromatin. The University of Connecticut station (Bruno, Koo) has been actively involved in identifying the bioactivities of green tea in experimental models of obesity-triggered nonalcoholic fatty liver disease (NAFLD). Of the major tea products, green tea is uniquely processed such that its rich polyphenolic content, consisting mainly of catechins, is preserved. Presumably, the in vitro reactive oxygen and nitrogen scavenging abilities of green tea catechins contribute to their bioactivity in human health, but their relatively low bioavailability and high biotransformation raises debate over their ability to optimize human health. Green tea extract (GTE) attenuated hepatic steatosis and hepatic injury in genetically obese (ob/ob) mice. Furthermore, GTE-mediated attenuation in hepatic steatosis was accompanied by decreased mRNA expression of adipose sterol regulatory element-binding protein-1c, fatty acid synthase, stearoyl CoA desaturase-1, and hormone sensitive lipase as well as decreased serum nonesterified fatty acid concentrations. Thus, GTE decreased the efflux of lipid from adipose to the liver where it would otherwise be esterified and stored as triglyceride. Parallel studies demonstrated that GTE decreased hepatic TNF-± protein and inhibited adipose TNF-± mRNA expression. Hepatic total glutathione, malondialdehyde and Mn- and Cu/Zn-superoxide dismutase activities in obese mice fed GTE were also normalized to the levels of lean littermates. Also, GTE increased hepatic catalase and glutathione peroxidase activities and these activities were inversely correlated with alanine aminotransferase (ALT) and liver lipids. Thus, GTE mitigated hepatic steatosis, injury, and oxidative stress by decreasing adipose TNF-±, lipogenic, and lipolytic gene expression, decreasing hepatic lipid peroxidation and TNF-± concentration, and enhancing hepatic antioxidant defenses. These findings suggest that GTE may be an effective dietary strategy to mitigate obesity-triggered NAFLD. Studies to identify the mechanisms by which GTE protects against liver injury, associated with inflammation, in a dietary-induced obese rodent model of NAFLD are currently underway. Wistar rats were fed a high fat diet containing 0, 1, or 2% GTE or a low-fat control diet containing no GTE for 8-wk. High-fat controls had greater (P<0.05) body and adipose mass. GTE lowered these to that of low-fat controls without affecting food intake. Also, GTE prevented obesity-mediated increases in insulin-resistance and serum leptin. Serum ALT and aspartate (AST) aminotransferase activities were 81-98% greater in rats fed a high-fat diet. GTE decreased their activities by 32-39%. GTE restored hepatic glutathione levels and prevented obesity-triggered increases in hepatic malondialdehyde. Livers from high-fat controls had greater mRNA and protein expression of MCP-1 and TNF-±. GTE decreased the mRNA expression of MCP-1 and TNF-± and their protein levels in liver. Hepatic MCP-1 protein was positively correlated with ALT, AST, and MDA and inversely correlated with hepatic glutathione, suggesting that GTE-mediated improvements in liver inflammation decrease hepatic injury and oxidative stress associated with NAFLD. These findings indicate that GTE protects against obesity-triggered NAFLD by decreasing hepatic inflammation and oxidative stress that otherwise lead to hepatic lipid peroxidation and hepatic injury. The incorporation of the tropical fruit, mango, into the diet is being investigated at Oklahoma State University (Lucas) to test effects of blood glucose and body fat accumulation. Although pharmacological options are available for the treatment of these risk factors for coronary vascular disease, alternative approaches that take advantage of the bioactive components in foods are highly desirable. A study compared the effect of mango to that of a hypocholesterolemic drug (fenofibrate) and a hypoglycemic drug (rosiglitazone) in reducing body fat and improving clinical parameters (i.e., blood glucose and lipid profile) of mice fed a high fat diet (HF). Male C57BL/6J mice were randomly divided into six treatment groups: control, HF, HF + 1% (w/w) mango or 10% mango (w/w), HF + fenofibrate (500 mg/kg diet), and HF + rosiglitazone (50 mg/kg diet). After eight weeks of treatment, both doses of mango prevented increases in visceral fat mass and % body fat due to high fat diet in a manner similar to fenofibrate and rosiglitazone. Supplementation with 1% mango was more effective than 10% mango, rosiglitazone, or fenofibrate in improving glucose tolerance. Additionally, mango reduced plasma glucose, total cholesterol and non-esterified fatty acids. Findings suggested that incorporation of mango in the diet lowered risk factors for CVD in this rodent model of high-fat diet-induced obesity. Evaluation of effects of food and beverage components on bone quality have continued at Oklahoma State University (Stoecker). Components evaluated include orange and ruby red grapefruit pulp as well as components of green tea. Studies of effects on rat bone of feeding carrots high in anthocyanins are continuing. These bones from small animal models of male and female osteoporosis are being analyzed for mass and density by dual energy X-ray absorptiometry (DEXA) and for bone microarchitecture by micro-computed tomography (µCT). Data generated by µCT are being incorporated into finite element analysis models to estimate effects of food components on breaking strength of bone. Purdue University (Weaver) has compared the effect of receiving dietary calcium from nonfat dry milk solids and calcium carbonate on bone health during growth and subsequent bone maintenance. Milk consumption is declining and calcium from fortified foods and supplements is more prominent, although after early puberty most females consume inadequate calcium. To address this problem female rats were fed a nutrient adequate diet, but the source of calcium was varied during growth (10 weeks). Some rats were maintained for an additional 10 weeks in a calcium inadequate diet. Bones of rats fed non fat dry milk solids during growth were longer, wider, more dense, and had greater peak breaking force than rats fed calcium carbonate. Furthermore, rats fed milk during growth were better protected from subsequent inadequate calcium diets. Purdue University (Weaver) also compared several botanical supplements containing isoflavones and traditional therapies for treating osteoporosis for their ability to suppress bone resorption. Several sources of botanical supplements have been marked as estrogen replacements for preventing menopausal bone loss. A novel Ca-41 method was used to compare supplements from two types of soy, red clover, and kudzu, estradiol, and a bisphosphohate in postmenopausal women. Only the soy supplements significantly suppressed bone turnover. The effect was modest compared to traditional therapies. Translation of science to the public remains a challenge and University of Illinois (Chapman-Novakofski) is investigating various mediating variables that influence behavior. Knowledge alone is not effective in changing behavior; thus, grounding in a behavioral theory is required for effective interventions targeting diet and lifestyle change. Online interactive education is being investigated as an effective alternative or augmentation to group education settings. Scientists in the W2002 group have collaborated on a number of research projects which range from genetic and epigenetic mechanisms by which nutrients and bioactive food components affect health and disease to translation of in vitro and animal studies on nutrients and bioactives to human populations. Strengths and limitations of available biomeasures are evaluated and methods for measurement of oxidative stress and/or inflammation as intermediate indicators of chronic disease risk are shared.