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W2002: Nutrient Bioavailability--Phytonutrients and Beyond

Annual/Termination Reports (SAES-422): [03/13/2009] [06/08/2010] [06/08/2011] [08/17/2012]

Date of Annual Report: 03/13/2009

Report Information:
  • Annual Meeting Dates: 01/15/09 to 01/16/09
  • Period the Report Covers: 10/2007 to 01/2009

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Copy of Minutes attached


    URL: Copy of minutes

    Accomplishments:
    Short Term Outcomes & Activities: This research group has had a highly productive year. First and foremost we were able to compete and secure funding as the W2002 Bioactive research group. This effort included a re-analysis of goals, objectives and strategic partnerships as well as the integration of several new members to enhance the group dynamics and capacity.

    The University of Arizona (Thomson) was awarded a USDA Bioactive Food Components grant to complete as controlled feeding trial to test the dose-response of vegetable intake in modulating oxidative stress and inflammatory response in overweight post-menopausal women. This study will identify not only changes in biomarkers of vegetable exposure but also assess the association of change in these biomarkers and change in oxidative stress and inflammation. The study is testing doses of 2, 5 and 10 servings of vegetables daily. Discussions with W2002 UConn researchers led to identification of optimal biomarkers to assess oxidative stress and inflammatory response for this research. AZ has also had the opportunity to collaborate with OSU researchers to support efforts to assess dietary exposure estimates to BAFC in cruciferous vegetables for two on-going studies. In addition the Thomson Clinical research group has published several manuscripts to support research in BAFC relevant to the W2002 group. In addition, an abstract presenting the a green tea and weight loss intervention trial among breast cancer survivors was presented at FASEB in 2008 and submitted January 2009 for peer-review.

    Collaborations are strong between Oregon State University and AZ as well as several other sites where Dr. Ho is advancing our understanding of the role of BAFC in cruciferous vegetables to reduce cancer risk in at-risk people. 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. These epigenetic alterations affect gene expression without directly changing DNA sequences, but rather turn on or off gene expression by post-translational modifications. Interestingly, many of these epigenetic modifications can also be modified by dietary factors. For example, pharmacological histone deacetylase (HDAC) inhibitors are currently being tested in human clinical trials and are proposed to have potent anti-cancer activity. The researchers have found that sulforaphane, a chemical found cruciferous vegetables is also an HDAC inhibitor, increases acetylated histone levels and has anti-cancer properties in the prostate. Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through other cancer chemopreventive mechanisms. The group recently reported on a novel mechanism of chemoprotection by SFN in human colon cancer cells and prostate epithelial cells, namely the inhibition of histone deacetylase (HDAC). In human subjects, a single dose of 68 g BroccoSprouts® inhibited HDAC activity significantly in peripheral mononuclear cells, 3 and 6 h following consumption. HPLC-mass spec methods have been developed for identifying SFN and its metabolites in plasma and urine. A significant increase in urinary and plasma SFN and metabolites can be found following broccoli sprout consumption 3-6 hours following consumption. We have also found that SFN specifically targets prostate cancer cells. Administration of sulforaphane selectively induces cell death in cancer cells, but not normal prostate epithelial cells. This work suggests that phytochemical may have the ability to alter epigenetic events that lead to disease prevention.

    Dr. Rich Bruno of U Conn has common research interest with several W2002 investigators related to the role of oxidative stress in chronic disease risk. Specifically he is investigating the role of oxidative stress and excess hepatic lipid accumulation which are strongly implicated in obesity-triggered nonalcoholic fatty liver disease (NAFLD). His research group has evaluated the protective bioactivities of green tea extract (GTE) in mitigating these NAFLD implicated events in genetically obese (ob/ob) mice. 6-Wk dietary supplementation of GTE at 1% (w/w), but not 0.5% reduced (p<0.05) hepatic triglyceride and total lipid whereas both doses reduced the mRNA levels of fatty acid synthase and stearoyl-CoA desaturase suggesting that GTE inhibits de novo lipogenesis. Concurrently, they observed GTE-mediated decreases in serum alanine aminotransferase, hepatic TNF-± and hepatic malondialdehyde. In addition, GTE increased the activities of the CuZn- and Mn-superoxide dismutase, catalase and glutathione peroxidase suggesting that GTE attenuated hepatic injury in association with improved oxidative stress and inflammation. Thiese works have resulted in four publications this year. These publications support the role for tocotrienols and dietary GTE in protecting against NAFLD by regulating hepatic de novo lipogenesis and attenuating inflammatory and oxidative stress responses that contribute to NAFLD. The results provide common research ground for Drs. Bruno and Thomson re: oxidative stress and disease risk as well as the evaluation of the efficacy and mechanisms of green tea in modulating obesity.

    Dr. Barbara J. Stoecker, Ph.D  Oklahoma as two lines of scientific investigation relevant to the W2002 efforts. The first is related to dietary components and cadmium-induced bone loss in ovariectomized rats Cadmium (Cd) has detrimental effects on bone; however, some dietary components may affect Cd toxicity. Er research group has examined the effects of dietary supplementation of potassium phosphate and/or dried plum (DP) on Cd-induced bone damage. Fifty, 90 day-old ovariectomized Sprague-Dawley rats were assigned to five treatments (n=10): 1) control (3gP/kg diet), 2) 50mg Cd and 3gP/kg, 3) 50mg Cd and 12gP/kg, 4) 200mg Cd and 3gP/kg, and 5) 200 mg Cd and 12gP/kg diet. After 45 days, half the rats in each treatment had 15% DP added to diets for 3 more months. Femoral cortical bone and microarchitecture of L4 vertebra trabecular bone was assessed with microcomputed tomography (µCT). Bone strength was evaluated using finite element analysis. Cortical thickness was decreased by Cd and by high P (p < 0.0001) but increased by DP (p<0.003). L4 bone volume fraction, trabecular separation and connectivity density were impaired synergistically by Cd and high P. With higher dietary Ca/P ratios, DP frequently maintained microarchitecture in Cd-50 rats but not in Cd-200 rats. Force to compress L4 trabecular bone was consistent in showing significant detrimental effects of Cd and high P and beneficial effects of DP in the Cd-0 and Cd-50 groups when high P was not fed. The other area of research interest of the Stroecker laboratory is in regards to the role of green tea polyphenols and bone turnover in rats. The effects of green tea polyphenols (GTP) on bone microarchitecture in middle-aged female rats without (sham, SH) and with ovariectomy (OVX) were evaluated to evaluate GTPs antioxidant capacity. A 16-week study was performed based on a 2 (SH vs. OVX) × 3 (no GTP, 0.1% GTP, and 0.5% GTP in drinking water) factorial design using 14-month-old female rats (n=10/group). An additional 10 rats were euthanized at the beginning of the study to provide baseline parameters. Analysis using dual-energy X-ray absorptiometry, histomorphometric, and micro-computed tomography [microCT in OSUs laboratory] showed that GTP supplementation resulted in (a) increased trabecular volume, thickness, number, and bone formation of proximal tibia, periosteal bone formation rate of tibia shaft, and cortical thickness and area of femur, and (b) decreased trabecular separation and bone erosion of proximal tibia, and endocortical bone eroded surface of tibia shaft. These results suggest that drinking water supplemented with GTP mitigated deterioration of bone microarchitecture in both intact and ovariectomized middle-aged female rats by suppressing bone erosion , enhancing bone formation, and modulating endocortical and cancellous bone compartments, resulting in a larger net bone volume. This line of research supports collaboration with Dr. Bruno (UConn), Dr. Thomson (UAz) and Dr. Weaver (Purdue). In addition, Dr. Stoeker has an active line of research related to nutrient adequacy during pregnancy and childhood in developing countries affording an opportunity for collaborative international research.

    Obesity is associated with an increased risk of colon cancer and this has been a focus of the research of Dr. Norman Hord. A dearth of nontumorigenic colon epithelial cell model systems are available to address reductionist hypotheses concerning the interaction of endocrine products of adipose tissue and cancer risk. We have used conditionally immortal murine colon epithelial cells with distinct adenomatous polyposis coli (Apc) genotypes (IMCE (Apc Min/+) and YAMC (Apc+/+) to address the roles of leptin, adiponectin and interleukin 6 (IL-6) on cell number homeostasis and to characterize interactions with secreted products of macrophages. We demonstrated that leptin, an adipose-derived hormone, induces cell proliferation in IMCE, but not YAMC, cells by inducing autocrine IL-6 production and trans-IL-6 signaling. Microarray analysis revealed that leptin-induced changes in genes regulating the Wnt/beta-catenin-mediated pathway including Mdm2, Pik3r1, and Rb1. Leptin induced IGF-mediated pathway gene expression changes and their protein products in IMCE cells. In the IMCE cells IGFBP-6, IGF-1, and Crim1 expression was upregulated, while IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and Nov expression was downregulated by leptin treatment. While leptin promoted the proliferation of IMCE cells, we have also shown that it induces the production of chemokines which may activate macrophages and promote macrophage cell chemotaxis. As such, these data provide evidence for leptin-induced cross-talk between IMCE cells and macrophages that model a potential promotional mechanism. Low serum adiponectin, a major secretory product of white adipose tissue, is associated with colon, prostate and breast cancer; serum adiponectin levels decrease as body mass index (and leptin) increase. Under serum-free conditions, adiponectin (1 µg/ml) inhibited leptin-induced autocrine IL-6 production, soluble IL-6 receptor shedding, trans-IL-6 signaling and subsequent STAT3 phosphorylation in IMCE cells. Adiponectin inhibited leptin-induced cell proliferation in the IMCE cells and this inhibition was associated with I kappa B-alpha phosphorylation, I kappa B-alpha degradation and decreased NF-kappaB p65 DNA activation and binding. These data indicate that adiponectin acts on preneoplastic colon epithelial cells to regulate cell growth via 2 distinct pathways inhibiting leptin-induced NF-kappaB-dependent autocrine IL-6 production and trans-IL-6 signaling.

    Edralin A. Lucas (Oklahoma) I studying Momordica charantia and the modulation of glucose and lipid parameters in mice fed high fat diet. A high fat diet contributes significantly in the development of obesity and diabetes, two major public health concerns in the US and worldwide. Momordica charantia (MC), also known as bitter melon, is a widely consumed vegetable in Asia and reported to have hypoglycemic properties. This study compared the effects of freeze-dried MC with the PPARg agonist- rosiglitazone (rosi), and the known PPARa agonist- fenofibrate (feno), on body weight and clinical parameters using a mouse model of diet-induced obesity. Eight wk old male C57BL/6 mice were randomized to 8 dietary treatment groups (n=20/group): control diet (ad lib), control diet (pair fed), high fat (HF) diet, HF + 1% MC (w/w), HF + 10% MC (w/w), HF+1% MC seeds (w/w), HF + rosi (50mg/kg diet), and HF + feno (500mg/kg diet) for 8 wks. Significant differences in body weights were observed within one week of beginning the experimental diet. The final body weights of mice receiving the 10% MC were similar to the mice receiving the control diet (ad lib). Rosiglitazone and 1% MC were not able to reduce body weight; however, fenofibrate and MC seeds mildly reduced body weight. HF fed mice exhibited the highest percent body fat. Interestingly, 10% MC prevented the increase in adiposity due to high fat diet. A high fat diet containing 10% MC, similar to rosiglitazone, normalized blood glucose after a glucose tolerance test. Triglycerides, total cholesterol, and glucose were all elevated due to high fat diet. The higher dose of MC modulated these clinical parameters similar to fenofibrate and rosiglitazone. Fenofibrate caused an enlargement of liver which was not observed in other treatment groups. The mechanism by which MC modulates glucose and body weight warrants further investigation.

    Dr. Janos Zempleni (University of Nebraska at Lincoln) has a focused research program in the area of repression of transposable elements by histone biotinylation and its role in cancer prevention. Transposable elements such as long terminal repeats (LTR) constitute about 45% of the human genome; transposition events impair genome stability. Fifty-four promoter-active retrotransposons have been identified in humans. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, preventing transposition events and abnormal regulation of genes. Here, we demonstrate that the covalent binding of the vitamin biotin to lysine-12 in histone H4 (H4K12bio) and lysine-9 in histone H2A (H2AK9bio), mediated by holocarboxylase synthetase (HCS), is an epigenetic mechanism to repress retrotransposon transcription in human and mouse cell lines and in primary cells from a human supplementation study. Abundance of H4K12bio and H2AK9bio at intact retrotransposons and a solitary LTR depended on biotin supply and HCS activity, and was inversely linked with the abundance of LTR transcripts. Knockdown of HCS in Drosophila enhances retrotransposition in the germline. Importantly, his research group has demonstrated that depletion of H4K12bio and H2AK9bio in biotin-deficient cells correlates with increased production of viral particles, transposition events, and ultimately decreases chromosomal stability. Collectively, this study reveals a novel diet-dependent epigenetic mechanism that could affect cancer risk. This work has and will continue to afford opportunities for collaboration with several other W2002 investigators working in cancer prevention research.

    Dr. Andy Clifford (UCDavis)as completed compelling research in the area of age-related macular degeneration (AMD), the most common cause of irreversible blindness in elderly Americans. There is now evidence that the carotenoids, lutein and zeaxanthin are key to protecting against AMD, by filtering out blue light at a pre-receptoral level, or by quenching free radicals. Lutein and zeaxanthin are dietary xanthophyll carotenoids, that are delivered to the retina via plasma lipoproteins. Mechanisms governing the selective retinal capture and accumulation of lutein and zeaxanthin, over other carotenoids, are unknown except that lipoproteins/apolipoproteins play a key role. Xanthophyll-binding proteins in the retina capture the xanthophyll carotenoids, the Pi isoform of GSTP1 is specific for zeaxanthin but the binding protein for retinal uptake of lutein remains elusive. To explore the beneficial effects of lutein and zeaxanthin for humans one must understand how they are absorbed from the intestine, transported in serum, and taken up by the retina, use of 14C accelerator mass spectrometry (AMS) and kinetic models of datasets to facilitate this task. Dr. Cliffords work has focused on improving high throughput biological/biomedical applications AMS to quantify minute amounts of 14C. The process involves oxidation of carbon (in sample of interest) to CO2 and then reduction of CO2 to graphite-like substances that coat a -400-mesh spherical iron powder (-400MSIP) catalyst. Prior AMS methods often failed to produce robust ion currents that are required for reliable, accurate, precise, and high-throughput AMS for biological/biomedical applications. Therefore, we described our optimized method for reduction of CO2 to high-quality uniform AMS targets (1) and characterized their physical (hardness/color), morphological (Scanning Electron Micrographs), and structural (Fourier Transformed-Infra Red, FTIR; Raman; and X-Ray Defraction, XRD spectra) characteristics that guarantee accurate, precise, and high-throughput AMS measurement (2). In addition the technology was used to determine how ingested 14C-all-trans [10,10',11,11'-14C]-b-carotene is absorbed from the intestine, transported in serum, and metabolized in vivo in humans. The group has demonstrated excentral cleavage of b-carotene in humans for the first time (3). Finally, we discovered that the apparent digestibility of the 14C dose was 50 % (4). The metabolic fecal elimination and AUC0- were 0.05 and 41 %, respectively. The portion of 14C dose eliminated in urine varied 8.2 %. Plasma 14C-b-carotene and 14C- REs accounted for most of the absorbed 14C. Feces was the major excretory path. These data suggested the variable elimination of 14C via urine accounted for the variable plasma/serum responses to ingested b-carotene reported in prior studies. Our data also suggested that plasma levels of ROH plus REs should be considered in estimating vitamin A equivalency of ingested b-carotene. These methodologies have strong potential for application to numerous BAFC and thus offer opportunities to several of the W2002 investigators for collaboration.

    Barry Shane University of California, Berkeley 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. We have continued to evaluate the B12-dependent methionine synthase (MS) and methylene-tetrahydrofolate reductase (MTHFR) genetic mouse models to mimic the effects of these polymorphisms and to evaluate their effects on metabolism and how this is modified by nutritional status. One carbon metabolic fluxes and DNA and histone methylation has been evaluated in these animals and in embryonic fibroblasts. We have 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. We continue to evaluate genetic risk factors for neural tube defects. We are continuing to identify putative modifier genes which influence folate status, homocysteine levels, and methylation potential using a number of mouse strains, and are evaluating the interaction between iron and folate status. We have measured myelination rates in the B12-deficient mouse and have shown that remyelination is impaired. Mathematical models are being developed to better understand regulatory aspects of one carbon metabolism. The impact of this work is significant in that neural tube defects are the most common birth defects in humans and identification of genetic risk factors for this condition will allow screening to identify at risk individuals. Polymorphisms in genes encoding folate-dependent enzymes have been implicated as risk factors for cancer and vascular disease. The studies may indicate whether this risk can be modified by dietary changes and may shed some insight into the etiology of vitamin B12 deficiency symptoms and interactions between folate and iron.

    Dr. de Mejia from the University of Illinois joined the W2002 investigative team this year as her research focuses on the molecular mechanisms of chemoprevention of bioactive food components, mainly proteins/petides and flavonoids, and their safety. Thus, opportunities for collaborations with several investigators will be abundant. Her research focuses on the study of food components with health benefits; analysis, characterization and mechanism of action of antimutagenic and anticarcinogenic compounds in foods (legumes, oilseeds, and vegetables). Currently the group is working with bioactive proteins in different legumes and investigates the role of processing on the presence, concentration and physicochemical characteristics of proteins/peptides with biological potential against chronic diseases as well as their safety such as allergenic potential. Similar to Bruno (UConn) and Thomson (UAZ) this research group is also studying the health benefits of tea, in particular the molecular mechanisms underlying the biological effects of ethnic teas used in folk medicine to combat several disorders including cancer. This scientific study will introduce new materials to improve human health. In 2007-2008 this research group published over 15 manuscripts indicating involvement of Dr. de Mejia is likely to result in even greater productivity for the W2002 group.

    Dr. Weaver at Purdue has accomplished instrumental work during this period evaluating bone mass in adolescents. She has found that about 25% of peak bone mass is acquired during the peak rate of bone acquisition in puberty, but factors which influence skeletal calcium accretion are not well understood. We evaluated the role of vitamin D status in calcium absorption and retention in adolescent white and black girls. Her research also evaluated predictors of calcium retention in adolescent boys. Data from controlled feeding studies on a range of calcium intakes were evaluated for factors which are thought to predict calcium utilization in 55 white and 55 black girls and 31 boys. In girls, calcium intake explained about as much of the variation in calcium retention as did race (12.3 vs. 13.7%, respectively). In boys, calcium intake and IGF-1 explained 21.7 and 11.5% of the variation in calcium retention. This shows lifestyle choices can be as important for skeletal growth as genetic determinants. This kind of analysis can only be derived from controlled feeding studies. This work has resulted in the publication of numerous peer-reviewed manuscripts (see publication list).

    The research of Dr. Winzerlings lab (UAz) is unusual in regards to the W2002 in that they work in mosquitoes as models for iron metabolism. They recently analyzed transferrin 2 from mosquitoes to determine whether this protein is likely to be involved in iron metabolism in this animal. Tf2 showed greatest identity to melanotransferrin from humans. However, like mammalian melanotransferrin, Tf2 in mosquitoes is unresponsive to exposure to iron in a blood meal as well as to iron administrated to mosquito CCL-125 cells in culture. Life stage analysis suggests that Tf2 is most likely involved in development. Ferritin is crucial to iron metabolism in mosquitoes and man. In mosquitoes it is a primary iron transport protein. We evaluated the ability of several chemical inhibitors to block ferritin secretion in mosquito cells. CI-976 a phospholipase inhibitor blocked secretion, whereas Brefeldin A did not, suggesting that ferritin is secreted from mosquito cells by budding of vesicles from the ER membrane. However, when fed to mosquitoes as part of a blood meal, CI-976 (in ethanol) failed to reduce egg numbers or increase mortality. Thus, we are using RNAi to evaluate the effects preventing iron transport from the blood meal to the ovaries and eggs. We also have initiated studies evaluating the ovarian and egg proteome expressed following an artificial blood meal with and without hemoglobin to female mosquitoes. In addition to our mosquito work, we collaborated with a team of scientists in the Czech Republic in work on iron metabolism in ticks that transmit disease. This work determined that Tf2 is not likely involved in iron metabolism. Although CI-976 inhibits ferritin secretion from cells; feeding the compound to animals failed to reduce egg numbers or increase mortality of mosquitoes. Dietary iron is highly mitogenic in mosquitoes and stimulates synthesis of several ovarian and egg proteins.

    The Medeiros lab is involved in two micronutrient studies. The first one focuses on copper deficiency and cardiac hypertrophy with emphasis on mitochondria and the second on the role of iron in bone. Copper and heart disease studies: During the last year, the group published a paper on the gene program that is up-regulated in mitochondrial biogenesis in hearts from copper deficient rats. We have demonstrated that PGC1-± transcripts and protein are robustly up-regulated in hearts from copper deficient rats. This protein is thought to be the master regulator of mitochondrial biogenesis. Previously we have demonstrated that other proteins and transcription factors involved in mitochondrial biogenesis are up-regulated. However, many of these markers depend on PGC1-±. These results in tandem with other papers we have published prove that all of the markers of mitochondrial biogenesis are upregulated in hearts from copper-deficient rats. This occurs despite the fact that there does not appear to be a shift in substrate utilization. In heart disease, the heart will often shift from fatty acids to glucose as a fuel substrate source to maintain ATP levels. Here we measured rate limiting enzymes in glycolysis and found that medium chain acyl dehydrogenase (rate limiting for beta oxidation of fatty acids), phosphofructose kinase (rate limiting for glycolysis), and Phosphoenolpyruvate carboxykinase (gluconeogenesis enzyme) did not differ by copper status. This suggests that the increase in mitochondria is able to keep up with energy demands of the heart as an adaptive process. Using proteinomics, we were able to determine changes in several proteins, all of which were secondary to heart damage found in the copper deficient heart. Future studies have now focused upon chaperone proteins of cardiac cells as a function of copper deficiency. Iron restriction and bone integrity: The second area deals with iron deficiency and bone integrity. We have published previously that animals fed iron restricted diets, as well as calcium restricted diets either singly or in combination with one another, have reduced bone strength as measured by Instron breakage. Using mico-CT analysis we have been able to demonstrate increased porosity of trabecular of bone from rats fed either calcium or iron restricted diets. These studies were done in collaboration with Dr. Stoecker of Oklahoma State University. A salient additional finding from several of our studies on this topic is an apparent decrease in mineralization of bones from iron restricted animals and also with an in vitro bone cell system using an iron chelator to generate an iron deficiency. The next logical step in this study is to collaborate with others on the multi-state project to assess calcium kinetics as affected by iron restriction, looking at both bone accretion and turnover. A second line of work needed is to determine if the condition is reversible with iron repletion. Finally, human epidemiological studies are needed to confirm the feasibility of iron as a factor in human bone health. Collaborations related to iron and bone health are underway and involve Drs. Medieros, Weaver and Winzerling.

    Important to all this research is the role of translational research that brings research findings to the consumer. To support the translation of science at this level we have engaged the expertise and experience of Dr. Karen Chapman-Novakofski a well-recognized expert in behavior research and epidemiological study. Various mediating variables influence behavior and identifying those variables continues to be a research interest in epidemiological studies. In addition, we know that knowledge alone is not effective in changing behavior. Grounding in a behavioral theory is required for effective interventions targeting diet and lifestyle change. In addition, in-person education is becoming costly in terms of time and effort for both the educator and learner. In this regards, online interactive education may prove to be an effective alternative to group education settings, or at least augment such efforts. Her research has resulted in several publications during the report period and offers an opportunity for several W2002 investigators to expand collaborations to include epidemiological and/or outcomes-based research.

    Outputs: As a whole the research group within W2002 has published over 50 peer-reviewed manuscripts in 2007-2008 impacting healthcare, research technology and methodology, consumer knowledge and the food supply. We have met our objectives to expand knowledge of bioavailability of nutrients as well as to assess specific bioactivity of select BAFC in the human diet with a particular emphasis on bioactivity biomarkers/indicators of bone health, cancer risk, cardiovascular disease risk and inflammatory disorders such as obesity. In addition we have explored the role of epigenetics in health and have bridged opportunities between epidemiologists and basic scientists in regard to testing hypotheses across the spectrum of nutrition science research. We have shared methodologies, technologies, expertise and research findings in a meaningful, collaborative and interactive way thus supporting the advancement of scientific knowledge regarding the role of BAFC and nutrients in optimizing human health.

    Milestones: Successful renewal of the W2002 Multistate project was accomplished. For 2009 we plan to continue to build productive collaborative research projects and joint peer-reviewed manuscripts. In addition we are planning a translational research symposium for the 2010 FASEB meeting. To this end we have engaged 5 different Research Interest groups within FASEB and have submitted our proposal titled, From field to consumer-the science & translation of bioactive food component research. The symposium would be co-directed by Norman Hord and Richard Bruno members of W2002 and all speakers are within our research group.

    Impact Statements:
    1. 1. Advancing our understanding of the role of bioactive food compounds in optimizing health is a central impetus for the development of collaborative research of the W2002. Specifically efforts to identify the appropriate dose and intake of whole foods and isolated bioactive compounds will advance our ability to test the efficacy in clinical trials including assessment of impact on fatty liver disease oxidative stress, and inflammation all biological responses associated with obesity.
    2. 2. Collaborative research will foster sharing of research techniques that validate requirements for nutrients and bioactive food compounds. Examining bone microstructure response to nutrient or bioactive compound exposure using sophisticated labeling techniques in controlled human feeding studies is critical as we establish optimal requirements for health. Nutrient balance studies will support the establishment of revised DRIs for nutrients and tea polyphenols, anthocyaninis, carotenoids and isothiocyanates for cancer risk reduction.
    3. 3. Our efforts to develop and disseminate the research methods as well as technology and expertise beyond the W2002 group will culminate with our proposed symposia to be presented at FASEB in 2010. This proposal has been submitted for consideration by the planning committee. This is a viable approach to translating science to others of mutual interest and to assure a broader and more efficient spread of research approaches in relation to BAFC and health.
    4. 4. Federal Grant funding (PI or Co-I and peer-reviewed) secured for the 2008 year include 12 USDA funded grants among 9 of the W2002 investigators as well as several NIH , CDC and industry funded grants that will continue to foster bioactive compound research
    Last Modified: 16-Mar-2009

    Date of Annual Report: 06/08/2010

    Report Information:
  • Annual Meeting Dates: 03/04/10 to 03/05/10
  • Period the Report Covers: 01/2009 to 02/2010

    • Participants:
    Brief Summary of Minutes of Annual Meeting:

    URL: Copy of minutes
    Accomplishments:
    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.

    Impact Statements:
    1. 1) Concerns have been raised about increased cancer risk and exacerbation of B12 deficiency by folate fortification. The models developed may indicate whether chronic disease risk can be modified by dietary changes and may shed some insight into possible adverse effects of folate fortification.
    2. By quantifying the dynamic and kinetic behavior of the metabolism of beta-carotene and its metabolites as it occurs in vivo in humans, a dietary requirement of beta-carotene or its metabolites to meet a health promoting function can be specified.
    3. Incidence of non-alcoholic fatty liver disease (NAFLD) has paralleled the ongoing obesity epidemic and no well established therapeutic options exist for patients with NAFLD beyond weight loss exist and such lifestyle modifications have a poor long-term success rate. Dietary strategies, such as green tea, may help in the prevention of NAFLD.
    4. Calcium consumption from milk is declining and calcium from fortified foods and supplements is more prominent. However, in rat studies, 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.
    Last Modified: 29-Jun-2010

    Date of Annual Report: 06/08/2011

    Report Information:
  • Annual Meeting Dates: 04/07/11 to 04/08/11
  • Period the Report Covers: 03/2010 to 03/2011

    • Participants:
    Brief Summary of Minutes of Annual Meeting:

    URL: Copy of minutes
    Accomplishments:
    The participants of this multi-state project have been highly productive during the past reporting period as evidenced by >50 peer-reviewed publications among attending participants and enhancement of collaborations between project members. Project objectives are listed below along with scholarly activities of the lead station.

    Objective 1): Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and other food components and their environmental and genetic determinants.

    Objective 2): Evaluate the bioactivity of nutrients and other food components in order to elucidate their underlying protective mechanisms.

    Colorado State University (Jairam Vanamala). We developed a systematic approach to screen storage and processing effects on bioactivity of plant products (colored potatoes) by focusing on (i) analysis of both storage and processing (baking and chipping) on phenolic content (TP), composition, sensory attributes and biological properties (anti-proliferation and pro-apoptosis); and (ii) investigation of consumer and producer preferences for specialty potato products. We have also collected extensive samples from pigs (n = 40) consuming a high-fat diet for 12 wk followed by supplementation with white vs. purple potatoes at two different doses (10% vs. 20% of diet) with an aim to determine the appropriate dose needed to suppress an inflammatory cascade. We demonstrated that post-harvest storage of colored potatoes elevated TP and anti-oxidant activity (AA), but suppressed anti-proliferative and pro-apoptotic properties against human colon cancer cell lines. Storage also altered the LC-MS metabolite profile of all potato varieties tested. These results suggest that it is critical to use both analytical and biological assays in conjunction to determine the post-harvest storage and processing effects on bioactivity of plant products. Chipping, but not baking, altered the metabolite profile. Chips had approximately 5-10 times less bioactivity compared to the baked or fresh potatoes. To study the effect of storage and inter-varietal differences on chipped and baked potato sensory attributes, 114 untrained panelists rated the 7 potato cultivars using a 9-point hedonic scale at 30 and 90 days of storage. In general, phenolic content was inversely related to sensory attributes. These results suggest that it is critical to consider sensory attributes along with bioactive compound profile in developing health food products. Consumers were also more willing to pay higher premium for colored potato products if they are educated on health benefits.

    Kansas State University (Denis Medeiros). We have ongoing work examining the impact of copper-deficiency on cardiac chaperone proteins. We have for years studied the mechanisms as to why copper deficiency leads to cardiac hypertrophy and cardiomyopathy. We have described pathology of mitochondria including the gene program responsible for mitochondrial biogenesis. During the past reporting period, we detailed the impact copper deficiency on cardiac chaperone proteins, which are integral for ferrying copper between cell compartments in which copper is used, such as the synthesis of copper containing enzymes. The copper chaperone proteins we evaluated as a result of copper deficiency include CCS, Sco1, Cox 17, and Ctr-1. Rats were fed diets either adequate or deficient in copper from weanling for 5 wk thereafter. Copper deficiency did not change Ctr-1 or Cox 17 expression. Ctr-1 is thought to regulate the amount of copper entering the myocyte or heart cell. We had expected that this protein would increase, but it did not. Cox 17 is involved in brining copper to the mitochondria. Sco1 and CCS were two chaperone proteins that increased in the heart as a result of copper deficiency. Sco1 delivers the copper to cytochrome C oxidase and CCS to the site of Cu, Zn superoxide dismutase synthesis. We also studied the impact of copper deficiency on cytochrome C oxidase subunits I and IV. We show that nuclear encoded subunits of cytochrome c oxidase were down regulated. In our earlier studies, we used a polyclonal antibody to formulate to this conclusion. We re-performed our study using mono-clonal antibodies, as other have used to determine if the source of antibodies could explain this discrepancy. Using mono-clonal antibodies we found that mitochondria encoded subunit I (Cox I) and the nuclear encoded subunit IV (Cox IV) were both significantly reduced, in contrast to what we previously reported. This suggests that copper deficiency may be causing a change in the mitochondrial encoded subunits of cytochrome c oxidase whereby the monoclonal antibodies are perhaps able to bind to the mature form of the protein and the polyclonal form may be binding to the precursor or immature form of the protein. During the past reporting period, our laboratory has also successfully completed an exhaustive review regarding the role of iron in bone metabolism. The information is now being utilized to develop a strategy on how to better translate the findings of animal models on a high risk human population and to determine if iron status is a predictor of lower bone mineral density, and stress fractures in particular, in humans.

    Michigan State University (Norm Hord). Dietary exposure to nitrates and nitrites is associated with cardiovascular health benefits and, in the context of processed meats consumption, gastrointestinal cancer risk. Our work describes the scientific basis for these risk assessments by examining the effect of nitrite on phenotypes associated with colorectal cancer (CRC) risk in human carcinoma cells. Nitrite and nitrate used exogenously in processed meats to enhance taste and microbial food safety is associated with increased cancer risk, but these compounds are also found in vegetables, and associated with lower cancer risk. To clarify this controversy, we determined whether nitrite promotes or blocks phenotypes associated with the promotion of cancer in colon epithelial cells. The effect of nitrite on proliferation and invasion of 4 different stages of colorectal epithelial cancer was examined. Nitrite has no effect on stage 1 SW1116 colon cancer cell at any concentrations; nitrite inhibits stage 2 SW480 proliferation at 10nm-100µM and inhibits stage 3 HCT15 at 100nm and 1µM, but promotes proliferation in stage 4 cells at 100µM. Nitrite also inhibits stage 3 SW480 cells invasion in a dose-dependent manner. However, it significantly promotes the invasion of stage 4 cells at the highest concentration. FACS data indicates that nitrite dose-dependently decreased cell cycle progression in SW480 and HCT15 with changed G2/M transition and delayed G1 phase entry. However, 100µM nitrite promoted cell cycle progression in COLO205 cells with increased S-phase entry. Taken together, nitrite can inhibit cancer cell progression at low doses and early stage but may promote cancer cell progression at higher doses in stage 4 colon cancer indicating a dose and stage dependent effect. Subsequent studies were performed to estimate exposure to nitrates and nitrites from human, bovine and formula milks relative to regulatory intake limits. The World Health Organization and American Academy of Pediatrics recommends exclusive consumption of human milk for the first 6 mo of life because of the nutritional and immunological benefits for the infant. Consumption of formula, bovine, and soy milk may be used as alternatives to human milk for infants. Human milk concentrations of colostrum (expressed d 13 postpartum; n1/412), transition milk (expressed d 37 postpartum; n1/417), and mature milk (expressed >7 d postpartum; n1/450) were 0.08 mg/100mL nitrite and 0.19 mg/100mL nitrate, 0.001mg/100mL nitrite and 0.52mg/100mL nitrate, and 0.001mg/100mL nitrite and 0.3 mg/100mL nitrate, respectively, revealing that the concentrations of these anions change as the composition of milk changes. When expressed as a percentage of the World Health Organizations Acceptable Daily Intake limits, Silk® Soy Vanilla (WhiteWave Foods, Broomfield, CO) intake could result in high nitrate intakes (104% of this standard), while intake of Bright Beginnings Soy Pediatric® formula (PBM Nutritionals, Georgia, VT) could result in the highest nitrite intakes (383% of this standard). The temporal relationship between the provision of nitrite in human milk and the development of commensal microbiota capable of reducing dietary nitrate to nitrite supports a hypothesis that humans are adapted to provide nitrite to the gastrointestinal tract from birth. These data also support the hypothesis that the high concentrations of breast milk nitrite and nitrate are evidence for a physiologic requirement to support gastrointestinal and immune homeostasis in the neonate.

    Montana State University (David Sands). High intake of starchy (high glycemic) grains, legumes and potatoes is one of the leading causes of obesity and heart disease. In response, MSU has recently released a new variety of pea that has very low branched starch and can be used for production of low glycemic index flour and food products. Additional low glycemic varieties of legumes can be developed. We are actively expanding our selection of low glycemic varieties to include spring, winter and durum wheats, legumes, and potatoes. Such low glycemic index crops could demand a premium in price at the farm gate. Similarly, we will continue to develop high omega-3 and high vitamin E camelina for both food and animal feed, because of its benefits to humans and animals. MSU biochemists and geneticists will follow nutritionally important genes through crosses, using recently developed and sophisticated molecular (but not transgenic) techniques, greatly reducing the time to improve crops. The resultant varieties will be tested for growth and productivity statewide at the MSU Agriculture Research Centers and the nutritional composition of the new varieties will be assessed. New research collaborations have also been identified with group members (Bruno/UConn; Vanamala/Colorado) to examine the potential bioactivity of specialty crops in physiologically relevant model systems.

    Ohio State University (Mark Failla) has been studying the impact of type of dietary fat on transport of carotenoids and vitamin E across the Caco-2 human intestinal cell line. B-carotene, lutein and ±-tocopherol transport into the basolateral compartment was increased to a greater extent when cells were exposed to micelles containing free fatty acids mimicking the fatty acyl composition of olive, canola and soybean oils compared to micelles with fatty acyl profile mimicking butter. The amounts of ²-carotene, lutein and ±-tocopherol transported into the basolateral compartment was highly correlated with the amounts of triacylglycerides and apoB secreted. The carotenoids were primarily present in chylomicrons and vitamin E was distributed in both chylomicron and VLDL fractions. These data suggest that dietary triacylglycerides rich in unsaturated fatty acids and particularly oleate promote the absorption of carotenoids and vitamin E by stimulating the assembly and secretion of chylomicrons. The bioaccessibility and absorption of the 24-carbon carotenoid norbixin was also investigated using the coupled in vitro digestion/Caco-2 cell model. Norbixin, an abundant carotenoid in annatto, is extensively used as a natural coloring agent by the food industry. Norbixin added to milk was highly stable during simulated digestion and bile salts enhanced partitioning into the bioaccessible fraction, suggesting delivery to the apical membrane of absorptive cells in micelles. Uptake of norbixin was proportional to apical content and both all trans and cis isomers were transported across the monolayer.

    Oklahoma State University. Work from Dr. Lucas demonstrates that mango exerts important bioactivities that may mitigate the risk of obesity. Mango (Mangifera indica L.) provides a number of bioactive compounds that may play a role in the attenuation of excess body fat. The effects of freeze-dried mango fruit on body composition were examined in two separate studies involving mice fed high fat diet and ovariectomized (ovx) mice. In the first study, male C57BL/6J mice were randomly divided into four treatment groups (n=8/group): control, high fat diet (HF), HF + 1% or 10% mango (w/w). After 8 wk of treatment, mice receiving the HF diet had significantly higher epididymal fat mass and % body fat. Both doses of mango prevented the increase in epididymal fat mass and % body fat due to HF diet. In the second study, female C57BL/6 mice sham-operated and ovx and were randomly assigned to receive control diet, 5% or 25% (w/w) mango diet (n=8/group). After 8 wk of treatment, ovx mice had higher % body fat in comparison to the sham group. Both doses of mango had lower % body fat in comparison to the ovx group but not quite to the level of the sham group. The mechanism by which mango exerts this positive effects on body composition is being actively investigated.

    Oregon State (Emily Ho) has been actively examining the role of epigenetic alterations in the risk for cancer and the extent to which bioactive nutrients such as sulforaphane, catechins and zinc regulate the mechanisms related to cancer and chronic disease development. In both animal models and humans, dietary zinc deficiency increases DNA damage in peripheral blood cells. Importantly, these functional changes precede any changes in plasma zinc levels. Zinc status is also compromised with age with ongoing studies demonstrating that zinc supplementation in older animals reverses age-related zinc deficiency and inhibits age-related immune defects and inflammatory processes. Preliminary evidence further suggests that zinc alters DNA methylation patterns and may be a novel mechanism by which zinc affects gene expression. Separate studies examining plant-derived phytochemicals from tea and cruciferous vegetables have provide evidence of the chemoprotective effects of diet. Indeed, studies show that sulforaphane, a chemical found cruciferous vegetables is an HDAC inhibitor, increases acetylated histone levels and has anti-cancer properties in the prostate. Sulforaphane is an isothiocyanate found in cruciferous vegetables such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through other cancer chemopreventive mechanisms. We recently reported on a novel mechanism of chemoprotection by sulforaphane in human colon cancer cells and prostate epithelial cells, namely the inhibition of histone deacetylase (HDAC). We have also found that sulforaphane specifically targets HDAC3 and HDAC6 in prostate cancer cells, but not normal cells. This work suggests that this phytochemical may have the ability to alter epigenetic events that otherwise lead to disease prevention. In human trials, we have directly compared the effects of the whole food (broccoli sprouts) to commercially available supplements. We have found a significant decrease in bioavailability and impact on HDACs with supplements compared to the whole food. We believe the decreases in activity are due to lack of myrosinase release of sulforaphane from its glucosinolate precursors in the supplement. We have also performed new studies examining the effects of tea catechins on autoimmune responses, which were completed in collaboration with Bruno (UConn). Regulatory T cells (Treg) are critical in maintaining immune tolerance and suppressing autoimmunity. The transcription factor Foxp3 serves as a master switch that controls the development and function of Treg. We have found that EGCG both in vitro and in vivo can induce Foxp3 and increase Treg frequencies to cause immunosuppression. The mechanisms may be related to demethylation of the Foxp3 promoter.

    Purdue University (Connie Weaver). During the past reporting period, we have conducted several studies in animal models and humans to investigate the effect of dietary constituents thought to be beneficial for bone health by either enhancing calcium absorption or suppressing bone resorption. In both growing rats and humans we tested the effect of fibers including GOS and FOS on calcium absorption. We continued our studies of the effects of phytoestrogens on preventing bone loss in an OVX rat model and the effect of equol producing status on bone resorption in response to soy in postmenopausal women.

    University of California-Berkeley (Barry Shane). We have continued studies on the metabolic and nutritional effects of common polymorphisms in human folate-related genes that have been shown to influence disease risk. We have continued to evaluate the B12-dependent methionine synthase (MS) and methylenetetrahydrofolate reductase (MTHFR) genetic mouse models to mimic the effects of these polymorphisms and to evaluate their effects on metabolism and how this is modified by nutritional status. We have developed a mouse model that mimics the clinical effects of human B12 and folate deficiency, and which will allow us to investigate potential adverse effects of high folate intake. We continue to evaluate genetic risk factors for neural tube defects and to identify putative modifier genes which influence folate status, homocysteine levels, and methylation potential using a number of mouse strains and a cohort of students at Trinity College, Dublin.

    University of Connecticut (Richard Bruno and Sung Koo). We have been actively investigating the hepatoprotective actions of green tea on liver injury associated with obesity-induced nonalcoholic steatohepatitis. We conducted a green tea extract (GTE) intervention study in obese rodents during the past reporting period. Wistar rats (16-wk old, n=63) were fed a low-fat (LF; 10% kcal) diet containing no GTE or a high-fat (HF; 60% kcal) diet containing 0, 1, or 2% GTE for 8 wks. We then examined whether GTE reduces inflammation associated with NASH by regulating COX-2. Rats fed HF diets developed liver steatosis and had greater (P<0.05) hepatic malondialdehyde (MDA) and serum alanine aminotransferase (ALT). GTE normalized the levels of hepatic lipid, MDA, and ALT to those of LF controls. The activity and protein expression of hepatic COX-2 were greater in HF fed controls. GTE normalized COX-2 activity and decreased its expression below the levels of LF controls. Arachidonic acid (AA) was elevated in the total lipid, phospholipid (PL), and free fatty acid (FFA) fractions of hepatic lipid extract. Although GTE reduced total AA by 6-8% (P<0.05), the concentrations of AA in the PL and FFA fractions remained unaffected as was cytosolic phospholipase A2 activity. HF increased cytochrome P450 2E1 mRNA expression, whereas GTE did not affect its expression. Thus, data suggest that GTE reduces dietary fat-induced hepatic inflammation and oxidative stress by decreasing the activity and expression of COX-2, without altering the flux of AA between membrane PL and FFA pools. Additional studies are warranted to define whether GTE suppresses COX-2 mediated prostaglandin generation. In separate studies, studies led by Dr. Bruno have examined the bioavailability and pharmacokinetics of quercetin in humans. We enrolled otherwise healthy obese men and postmenopausal women (n = 4M/5F; 55.9 ± 6.4 y; 30.8 ± 4.1 kg/m2) to complete a randomized, cross-over study. Participants ingested quercetin (1095 mg) on 3 occasions with a fat-free (<0.5 g), low-fat (4 g), or high-fat (15 g) meal. Plasma was obtained prior to (0 h) and after quercetin ingestion (1, 2, 3, 4, 6, 8, 10, 12, and 24 h) to measure quercetin and its methylated metabolites isorhamnetin and tamarixetin using HPLC-Coularray. Compared to the fat-free trial, quercetin bioavailability increased by 32% (AUC0-24 h; P<0.05) during the high-fat trial only. The high-fat meal also increased maximum quercetin concentrations (Cmax; 1.60 ± 0.88 ¼M) by 45%, but time to maximum concentration (Tmax; 353 ± 105 min) and half-life (t1/2; 515 ± 144 min) were unaffected. Isorhamnetin AUC0-24 h increased by 19% and Cmax increased by 40% (0.24 ± 0.11 vs. 0.17 ± 0.06 ¼M) without affecting Tmax (486 ± 113 min) or t1/2 (1154 ± 633 min). Tamarixetin AUC0-24 h increased by 43% and Cmax by 46% (0.52 ± 0.29 vs. 0.36 ± 0.11 ¼M) without affecting Tmax (399 ± 110 min) or t1/2 (299 ± 287 min). Thus, dietary fat improves quercetin bioavailability, and subsequent biotransformation, by increasing its absorption. Further work is warranted to define the optimal amount and type of fat to improve quercetin bioavailability and the extent to which the bioactivity of quercetin is exerted through its methylated metabolites.

    University of Nebraska (Janos Zempleni) has been examining the biotinylation of histones mediated by holocarboxylase synthetase (HCS), which is an important mechanism to repress retrotransposons. De-repression of retrotransposons impairs genome stability and increases cancer risk. In previous studies we showed that both biotin and HCS deficiency decreases the enrichment of biotinylated histones at retrotransposons, leading to increased transcription of transposons, increased frequency of retrotranspositions, and increased incidence of chromosomal abnormalities. In these previous studies we also demonstrated that histone biotinylation marks co-localize with methylated cytosines and lysine-9-dimethylated histone H3 (H3K9me2), suggesting epigenomic synergies among these chromatin marks. We tested the hypothesis that the epigenomic synergies are mediated by physical interactions among HCS, the DNA methyl transferase DNMT1, the methyl-CpG-binding protein 2 (MeCP2), and the histone H3 methyltransferase EHMT-1. Analysis by yeast-two-hybrid assays, co-immunoprecipitation, and limited proteolysis assays are consistent with the theory that HCS interacts with DNMT1, MeCP2, and EHMT-1, and that these interactions might be responsible for the roles of biotin and methyl donors such as folate in genome stability and gene regulation. The common interests in epigenetic modification by dietary constituents is an area that continues to be examined in collaboration with Ho (Oregon State).

    Impact Statements:
    1. Wheats, potatoes, legumes and oil crops (such as Camelina) can all be selected for enhanced nutritional properties. This shift away from commodity and yield driven agriculture will lead to more profitability and sustainability of our agricultural sector. The health benefits to consumers will be considerable.
    2. Using bioassays in conjunction with analytical assays is critically important to assess storage/processing effects on the health beneficial properties of plant products. These approaches demonstrate that purple potatoes have greater anti-cancer activity compared to white/yellow potatoes and that baking is a better processing method to retain the biological activity.
    3. Our work has led to strategies that may lower cancer risk. We show that: i) biotin and folate synergize to maintain genome stability, thereby decreasing the risk of cancer and birth defects, ii) nutritional strategies that decrease oxidative stress, inflammation, DNA damage and/or target aberrant epigenetic alterations in prostate cancer may reduce the incidence of prostate cancer and associated health care costs, and iii) estimates of human exposure to dietary nitrates/nitrites will enable our understanding of these compounds in the etiology of colorectal carcinogenesis.
    4. At the level of nutrient bioavailability, our works have the potential to impact the development of chronic diseases associated with aging. We show that i) the quantity and quality of dietary fat are important determinants of carotenoid bioavailability and potential mediators of the bioactivity of carotenoids, ii) select prebiotic enhances intestinal absorbtion of calcium, and iii) absorption of the polyphenol quercetin is enhanced by small amounts of dietary fat and its greater bioavailability leads to greater biotransformation to its methylated metabolites.
    5. Our work examining the hepatoprotective actions of functional foods demonstrates that i) mango improves body composition in animal models of obesity, and could lead to an economical and safe option for reducing obesity in humans, and ii) green tea extract, in obese models, mitigates nonalcoholic steatohepatitis by decreasing hepatic inflammation, oxidative stress, and lipid accumulation. These works may help growers and food manufacturers identify novels uses of these plants in foodstuffs and the development of products that may reduce the growing trends of obesity.
    6. Polymorphisms in genes encoding folate-dependent enzymes have been implicated as risk factors for cancer and vascular disease. Neural tube defects are the most common birth defects in humans and identification of genetic risk factors for this condition will allow screening to identify at risk individuals. Our work strongly suggests that folate fortification has no adverse effects on vitamin B12 status, at least in a young human population, and our work in transgenic models have allowed a mechanistic study of the potential interactions between vitamin B12 and folate.
    Last Modified: 09-Jun-2011

    Date of Annual Report: 08/17/2012

    Report Information:
  • Annual Meeting Dates: 06/04/12 to 06/05/12
  • Period the Report Covers: 03/2011 to 03/2012

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Participants: Meeting Host - Jairam Vanamala (jairam.vanamala@colostate.edu)- Colorado State University

    Administrative Advisor - Tammy Bray (tammy.bray@oregonstate.edu) - Oregon State University

    Connie Weaver (weavercm@purdue.edu) - Purdue University

    Barbara Stoecker (barbara.stoecker@okstate.edu) - Oklahoma State University

    Barry Shane (bandie@berkeley.edu) - University of California-Berkeley

    Mark Failla (MFailla@ehe.osu.edu) - The Ohio State University

    Janos Zempleni (JZEMPLENI2@unl.edu) - University of Nebraska-Lincoln

    Emily Ho (emily.ho@oregonstate.edu) - Oregon State University

    Brian Lindshield (blindsh@k-state.edu) - Kansas State University

    Jennifer Teske (teskeja@email.arizona.edu) - University of Arizona

    Absent: Richard Bruno (Univ. of Connecticut; Edralin A. Lucas (OK State); Elvira de Mejia (U IL)

    Meeting was called to order at 8:30 AM.

    Welcome and Introductions:

    Participants were welcomed by the host and Colorado State University Administrators: Jairam Vanamala, Chris Melby, Department Head, Food Science and Human Nutrition; Dr. Jeff McCubbin - Dean, College of Applied Human Sciences; Craig Beyrouty, Dean, College of Agricultural Sciences.

    Dr. Vanamala and the administrators shared insights on value of transdisciplinary approach in addressing complex food and nutrition problems. Investigators introduced themselves and their programs.

    Tommy Bray spoke about brief history of W2002 and expectations for the multistate research meetings/participants. She answered questions from the audience regarding deadlines and processes for multistate renewal grant proposal.

    Presentations, June 4 Station reports were shared from each investigator in the following order:

    1) Connie Weaver - Discussed the effects of galactooligosaccharide (GOS) on colonic calcium absorption in pre-menarcheal girls and the rapid screening method accelerator mass spectrometry for tracer quantification; Indicated that the Diet Impact on Bone is Greatest During Periods of High Bone Turnover

    2) Janos Zempleni - Discussed the importance of Holocarboxylase Synthetase in Biotinylation and how grape compounds inhibits the Holocarboxylase Synthetase

    3) Emily Ho - Discussed the pharmacokinetics and bioavailability of sulforaphane and its chemoprotective role in modulating epigenetic events

    4) Mark Failla - Discussed oral metabolism of anthocyanins

    5) Barry Shane - Discussed B-vitamins, especially B12 and folate, in relation to 1-carbon metabolism

    6) Brian Lindsheild - Discussed the importance of 5alpha-reductase inhibitors in development and progression of prostate cancer

    7) Jennifer Teske - Discussed the role of sleep in chronic disease development

    Presentations, June 5

    Dr. Tammy Bray discussed the status of the W2002, its funding cycle, and indicated that the group should begin organizing for the renewal in the upcoming years.

    Additional station reports were shared from each investigator in the following order

    1) Barbara Stoecker - Discussed ongoing nutritional studies in international populations

    2) Jairam Vanamala - Discussed the in vivo anti-oxidant and anti-inflammatory activity of processed (baked vs. chipped) purple-fleshed potatoes; Presented the importance of utilizing in vitro and in vivo models in conjunction with analytical techniques in assessing the farm to fork operation on health-benefiting properties of plant foods

    Janos Zempleni was elected by group members to be administrative leader for the next annual cycle. Dr. Zempleni will coordinate next year's W2002 meeting in Nebraska. Objectives, timeline, and responsibilities for project renewal were discussed and decided that Dr. Zempleni, Ho and Vanamala will lead the efforts. Discussed the possibility of future collaborations among the participants and propsed to start a collaborative study on health-benefiting properties of color-fleshed potato anthocyanins.


    URL: Copy of minutes

    Accomplishments:
    The participants of this multi-state project have been highly productive during the past reporting period as evidenced by >50 peer-reviewed publications among attending participants and enhancement of collaborations between project members. Project objectives are listed below along with scholarly activities of the lead station.

    Objective 1): Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and other food components and their environmental and genetic determinants.

    Objective 2): Evaluate the bioactivity of nutrients and other food components in order to elucidate their underlying protective mechanisms.

    Colorado State University (Jairam Vanamala). Potato (Solanum tubersom) is the third largest source of phenolic compounds in the human diet after oranges and apples. We have shown that locally-grown purple fleshed potatoes are a greater source of bioactive compounds, even after prolonged storage and processing (baking chipping), retains in vitro anti-cancer properties, compared to white-and yellow-fleshed cultivars. These results suggest that colored potatoes are a potential healthier alternative to common potato cultivars. This year we focused on assessing anti-oxidant and anti-inflammatory properties of colored potatoes in vivo using obese pigs, an agriculturally important animal with similar nutritional metabolism to humans, as a model. Samples from 40 pigs consuming a high-fat diet for 12 weeks followed by supplementation with white vs. purple potato chips (10% vs. 20% of diet;5 weeks) were utilized to determine the effect of dose on oxidative and/or inflammatory markers. The potato diets had no effect on the food intake, weight gain and back-fat thickness. However, serum oxidative stress markers, 8-isoprostane and malondialdehyde levels, were lower in pigs consuming purple potato diet (10 %) compared to control or white potato diet. Mesenteric fat free fatty acid analysis revealed that both purple and white potato diet (10 %, Purple > White) had lower saturated fatty acids (SFA) and greater poly unsaturated fatty acids (PUFA) levels compared to the pigs consuming high-fat control. Correspondingly, serum TNF-á, a pro-inflammatory cytokine, was also suppressed by the potato diets compared to high-fat control. These results suggest that purple potatoes, even after processing, might suppress both oxidative stress/inflammatory markers in vivo via alterations in the fatty acid composition. We also completed extensive sample collection from 64 pigs (3 weeks post weaning) consuming high fat diet supplemented with (10 %) purple and white potatoes (raw, baked and chipped) for 13 weeks. An additional control (fed with low fat diet) was included to establish baseline levels of oxidative stress/inflammatory markers.

    University of California, Berkeley (Barry Shane). We have continued studies on the metabolic and nutritional effects of common polymorphisms in human folate-related genes that have been shown to influence disease risk. We have continued to evaluate the B12-dependent methionine synthase (MS) and methylene-tetrahydrofolate reductase (MTHFR) genetic mouse models to mimic the effects of these polymorphisms and to evaluate their effects on metabolism and how this is modified by nutritional status. We have developed a mouse model that mimics the clinical effects of human B12 and folate deficiency, and which will allow us to investigate potential adverse effects of high folate intake. We continue to evaluate genetic risk factors for neural tube defects and to identify putative modifier genes which influence folate status, homocysteine levels, and methylation potential using a number of mouse strains and a cohort of students at Trinity College, Dublin. Impact: Neural tube defects are the most common birth defects in humans and identification of genetic risk factors for this condition will allow screening to identify at risk individuals. Polymorphisms in genes encoding folate-dependent enzymes have been implicated as risk factors for cancer and vascular disease. Recently, concerns have been raised about increased cancer risk and exacerbation of B12 deficiency by folate fortification. The models we have developed may indicate whether chronic disease risk can be modified by dietary changes and may shed some insight into possible adverse effects of folate fortification. Although it has been suggested that folate fortification exacerbates vitamin B12 deficiency symptoms, our recent studies on the Trinity Student cohort do not support any adverse effects of folate fortification on vitamin B12 status in a young population. Our genetic studies may suggest novel biomarkers for assessing vitamin status.

    Purdue University (Connie M. Weaver). Two novel fibers, GOS and Soluble Corn Fiber, increased calcium absorption in pubertal children associated with increases in proportion of gut bifido bacteria. Equol producing capability determined through pre-screening did not affect the bone resorption attenuation response to soy consumption in postmenopausal women. Several plant extracts were effective in reducing net bone turnover in an OVX rat model.

    Oregon State University (Emily Ho). 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. The lab has focused on the examination of the interaction of bioactive nutrients such as sulforaphane, catechins and zinc on mechanisms related to cancer and chronic disease development.

    Zinc and chronic disease: In both animal models and humans, we have found that dietary zinc deficiency increases DNA damage in peripheral blood cells. Importantly, these functional changes precede any changes in plasma zinc levels. More recently we have found that zinc status is compromised with age. Zinc supplementation in older animals reverses age-related zinc deficiency and inhibits age-related immune defects and inflammatory processes. We have also have preliminary data that suggests that zinc alters DNA methylation patterns and may be a novel mechanism by which zinc affects gene expression and the inflammatory response.

    Plant-derived phytochemicals from tea and cruciferous vegetables: We have found that sulforaphane, a chemical found cruciferous vegetables is an inhibitor of histone deacetylases, increases acetylated histone levels and has anti-cancer properties in the prostate. We recently reported that SFN also causes decreases in DNA methyltransferase expression and causes hypomethylation of cyclinD2, a commonly repressed and hypermethylated gene in prostate cancer cells. Other phytochemicals derived from cruciferous vegetables, such as indole-3-carbinol may also have epigenetic targets and inhibit HDAC. This work suggests that phytochemical may have the ability to alter epigenetic events that lead to disease prevention. In human supplementation trials, we have directly compared the effects of the whole food(broccoli sprouts) to commercially available supplements. We have found a significant decrease in bioavailability and impact on HDACs with supplements compared to the whole food. Surprisingly, even when supplements are pre-treated with myrosinase, the release of sulforaphane from its glucosinolate precursors in the supplement is limited.

    Kansas State University (Brian L. Lindsheild). We're currently conducting a study to determine the effects of providing the 5alpha-reductase inhibitors finasteride and dutasteride starting at two different times on the development and progression of prostate cancer TRAMP mice. We're also using gas chromatography to quantify and characterize the fatty acids in saw palmetto supplements. Large clinical trials have found that both finasteride and dutasteride reduced the risk of prostate cancer; however they also increased the occurrence of more advanced prostate cancer among those who did develop it. Our research will provide evidence to help clarify finasteride and dutasteride are effective and provide information on whether these should potentially be recommended for men at high risk of prostate cancer. The saw palmetto project should provide information about what supplements contain and whether they're efficacious.

    The Ohio State University (Mark Failla). The amount of ²-carotene delivered to the plate after processing cassava according to traditional styles of cooking and ²-carotene bioaccessibility increased in proportion to provitamin A content in transgenic high ²-carotene cultivars. ²-carotene content during storage and its bioaccessibility were greater for transgenic potato containing the OR carotenoid storage protein than that for control potato. The bioavailability of carotenoids from a vegetable salad was increased in response to the amount of co-consumed fat with a trend towards increased carotenoid absorption when the relative amount of unsaturated fat was elevated. Xanthones from a mangosteen juice were identified as the free compounds and their phase 2 metabolites in serum and urine in healthy human participants.

    University of Nebraska at Lincoln (Janos Zempleni). Covalent binding of biotin to histones is catalyzed by holocarboxylase synthetase (HLCS) and has been linked with the repression of genes and repeat regions in human chromatin. However, less than 0.001% of histones H3 and H4 are biotinylated, thereby raising concerns that the abundance might be too low to elicit the strong phenotypes observed in HLCS- and biotin-deficient organisms in vivo. We are proposing an alternative model that integrates the findings from previous research by us and others. In this novel model, HLCS acts as an integral part of a multiprotein gene repression complex in human chromatin, and biotinylation of histones is a mere side effect of HLCS being in close physical proximity to histones. Specifically, we are proposing that HLCS interacts physically with the following chromatin proteins that are known to mediate gene repression by creating or interpreting epigenetic gene repression marks: euchromatic H3K9 methyltransferase EHMT-1, the maintenance DNA methyltransferase DNMT1, the methyl-CpG-binding domain protein MeCP2, histone deacetylases (HDACs) 1, 2, 3, and 8, the nuclear co-repressor N-CoR, and heterochromatin protein (HP) 1. Protein-protein interactions were predicted by using a new search algorithm and verified by using co-immunoprecipitation assays, limited proteolysis assays, and yeast-two-hybrid assays. As of today, we have confirmed physical interactions between HLCS and DNMT1, MeCP2, EHMT-1, and HDAC1. Studies with N-CoR and HP1 are in progress. Studies with synthetic inhibitors and transgenic models suggest that DNA methylation is a primary loading factor, followed by docking of HLCS, followed by docking of EHMT-1 and/or HDACs. Importantly, we discovered a novel biotinylation site in EHMT-1, namely K161, which might be essential for chromatin positioning of EHMT-1 and the subsequent methylation of K9 in histone H3. Impact. Biotin and folate synergize to maintain genome stability, thereby decreasing cancer risk and the risk for birth defects.

    Oklahoma State University (Barbara Stoecker). A study of 41 women (over 50 years of age) with and without metabolic syndrome (MetS) indicated that in addition to differences defining MetS, mean serum adiponectin was lower in the MetS group, and circulating insulin was higher (p<0.003). Insulin resistance was significantly elevated in the MetS group as was the android:gynoid fat ratio. Android fat was positively associated with serum glucose, triacylglycerols, and insulin and with insulin resistance, whereas gynoid fat showed no significant association with these variables. Total dietary antioxidant capacity was positively correlated with serum HDL-cholesterol, adiponectin and dietary fiber intakes which supports the idea that fruits and vegetables are good sources of antioxidants.

    Oklahoma State University (Edralin A. Lucas). Bitter melon (Momordica charantia, MC) has been shown to improve markers of insulin resistance including adiposity, glucose and lipid profile in mice fed high fat diet. Fatty acids can activate toll-like receptor (TLR) 4 signaling pathway resulting in the enhanced production and secretion of proinflammatory cytokines which may contribute to insulin resistance associated with diet induced obesity (DIO). We examined the effect of MC on glucose and lipid homeostasis in an animal model of DIO and the role of TLR4 in mediating these effects. Eight week old male TLR4 mutant (C3H/HeJ) and control (C57BL/6) mice were randomly assigned to four dietary treatment groups for eight weeks (n=12-13/group): control (10% calories from fat), high fat (HF, 60% calories from fat), HF+1% (w/w) MC, and HF+10% (w/w) MC. Our preliminary data suggests that the C3H/HeJ strain exhibited significantly higher body weight, body fat, plasma cholesterol, and triglycerides in response to a high fat diet when compared to the C57BL/6 strain regardless of dietary treatment. However, C3H/HeJ strain had significantly lower area under the curve after a glucose tolerance test, plasma fructosamine and free fatty acid in comparison to the C57BL/6 strain. Mice fed the 10% MC, but not the 1% MC, improved glucose tolerance in the C57BL/6 but not the C3H/HeJ strain, suggesting MC may act through TLR4 signaling to modulate glucose homeostasis. The effects of MC and TLR4 mutation on mRNA expression of genes involved in glucose and lipid metabolism are being assessed.

    University of Connecticut (Richard Bruno). Epidemiological observations suggest that the presence of nonalcoholic fatty liver disease (NAFLD) dramatically increases the risk for cardiovascular disease (CVD). Thus, we have conducted studies to examine the extent to which phytonutrients regulate oxidative stress and inflammatory responses in experimental and clinical models of NAFLD and CVD. In a high-fat feeding model of NAFLD, we examined the extent to which green tea extract (GTE) protects against liver injury by regulating hepatic and adipose inflammatory responses under the transcriptional control of nuclear factor kappa B (NFkB). Wistar rats (16-wk old, n=63) were fed a low-fat (LF; 10% kcal) diet containing no GTE or a high-fat (HF; 60% kcal) diet containing 0, 1, or 2% GTE for 8 wks. We then examined whether GTE reduced NFkB activation and expression levels of inflammatory mediators that are regulated in a NFkB-dependent manner. In a separate clinical study, a randomized cross-over trial was conducted in healthy men to examine the extent to which gamma-tocopherol (g-T) supplementation protects against vascular dysfunction otherwise induced by acute hyperglycemia by regulating lipid peroxidation and the ratio of asymmetric dimethylarginine relative to arginine (ADMA/Arg), an index of nitric oxide bioavailability. IMPACT: The findings of our NAFLD study in rats indicated that HF feeding increased serum alanine (ALT) and aspartate aminotransferases and hepatic lipids compared to the LF group. GTE at 1 and 2% decreased ALT and hepatic lipid relative to the HF group. In liver and epididymal adipose, the HF group had lower glutathione as well as greater mRNA and protein expression of TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) and NFkB binding activity than the LF group. Compared to the HF group, GTE at 2% increased glutathione and lowered protein and mRNA levels of inflammatory cytokines in both tissues. NFkB binding activities at liver and adipose were also lower, likely by inhibiting the phosphorylation of inhibitor of NFkB. NFkB binding activities in liver and adipose were correlated with ALT, and hepatic NFkB binding activity was inversely related to liver glutathione. These results suggest that GTE-mediated improvements in glutathione status are associated with the inhibition of hepatic and adipose inflammatory responses mediated by NFkB, thereby protecting against NASH. In our clinical study, healthy men completed a randomized, crossover study where they followed their usual diet or were provided g-T (500 mg/d, 5 d) prior to a fasting 75 g oral glucose challenge. Brachial artery flow-mediated dilation (FMD), plasma glucose, insulin, antioxidants, malondialdehyde (MDA), inflammatory proteins, Arg, and ADMA were measured at regular intervals during a 3 h postprandial period. g-T supplementation increased plasma g-T by 3-fold and its physiological metabolite, g-carboxyethyl-hydroxychroman by >9-fold without affecting glucose, arginine or ADMA. Baseline FMD responses, MDA, Arg, and ADMA were unaffected by g-T supplementation. Postprandial FMD decreased 30-44% following glucose ingestion, but was maintained during the g-T trial. g-T supplementation also attenuated postprandial increases in MDA that occurred following glucose ingestion. Plasma Arg decreased in both trials to a similar extent regardless of g-T supplementation. However, the ratio of ADMA/Arg increased time-dependently in both trials, but to a lesser extent following g-T supplementation. Inflammatory cytokines and cellular adhesion molecules were unaffected by glucose ingestion or g-T supplementation. Collectively, these findings suggest that short-term g-T supplementation maintained vascular function during postprandial hyperglycemia by attenuating lipid peroxidation and disruptions in nitric oxide homeostasis, independent of inflammation.

    Impact Statements:
    1. In general, phenolic content was inversely related to sensory attributes. These results suggest that it is critical to consider sensory attributes along with bioactive compound profile in developing health food products. Consumers were also more willing to pay higher premium for colored potato products if they are educated on health benefits.
    2. Neural tube defects are the most common birth defects in humans and identification of genetic risk factors for this condition will allow screening to identify at risk individuals. Polymorphisms in genes encoding folate-dependent enzymes have been implicated as risk factors for cancer and vascular disease. Recently, concerns have been raised about increased cancer risk and exacerbation of B12 deficiency by folate fortification. The models we have developed may indicate whether chronic disease risk can be modified by dietary changes and may shed some insight into possible adverse effects of folate fortification. Although it has been suggested that folate fortification exacerbates vitamin B12 deficiency symptoms, our recent studies on the Trinity Student cohort do not support any adverse effects of folate fortification on vitamin B12 status in a young population. Our genetic studies may suggest novel biomarkers for assessing vitamin status.
    3. Two novel fibers, GOS and Soluble Corn Fiber, increased calcium absorption in pubertal children associated with increases in proportion of gut bifido bacteria. Equol producing capability determined through pre-screening did not affect the bone resorption attenuation response to soy consumption in postmenopausal women. Several plant extracts were effective in reducing net bone turnover in an OVX rat model.
    4. Our work examining the hepatoprotective actions of functional foods demonstrates that i) mango improves body composition in animal models of obesity, and could lead to an economical and safe option for reducing obesity in humans, and ii) green tea extract, in obese models, mitigates nonalcoholic steatohepatitis by decreasing hepatic inflammation, oxidative stress, and lipid accumulation. These works may help growers and food manufacturers identify novels uses of these plants in foodstuffs and the development of products that may reduce the growing trends of obesity.
    5. Polymorphisms in genes encoding folate-dependent enzymes have been implicated as risk factors for cancer and vascular disease. Neural tube defects are the most common birth defects in humans and identification of genetic risk factors for this condition will allow screening to identify at risk individuals. Our work strongly suggests that folate fortification has no adverse effects on vitamin B12 status, at least in a young human population, and our work in transgenic models have allowed a mechanistic study of the potential interactions between vitamin B12 and folate.
    Last Modified: 18-Oct-2012
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