2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Improving the diet by increasing the consumption of whole grains, fruits and vegetables may decrease the incidence of cancer by 30-40%. Although fiber, vitamins and phytochemicals have received the most attention as chemopreventive components of a diet rich in grains, fruits and vegetables, minerals also may be important chemopreventive components. For example, human epidemiologic and supplementation studies, as well as extensive animal studies, have shown the efficacy of selenium in cancer prevention. Food contains different chemical forms of selenium as well as other dietary constituents which will influence the chemopreventive effect of selenium. Furthermore, recent studies suggest that dietary copper protects against colon cancer in several animal models. Other dietary minerals may be beneficial but their role in cancer prevention has not been thoroughly investigated. Mammary, colon and prostate cancers are the main types of cancer which are influenced by dietary factors. A key to understanding the relationship between optimal mineral intake and cancer is determining the effects of mineral intake on cellular processes such as gene expression, oxidative stress, apoptosis and signal transduction. Studies are and will be conducted to determine whether mineral elements such as selenium, copper, and zinc affect biomarkers of carcinogenesis, including carcinogen-induced aberrant crypt formation (a preneoplastic lesion for colon cancer), carcinogen-DNA adduct formation, oxidative status, selenoprotein and detoxifying enzyme activities, DNA methylation and tumor development. Min (multiple intestinal neoplasia) mice will be used to study the effects of trace minerals on the pathogenesis of intestinal cancer in a genetic model for cancer susceptibility. These mice contain a mutation in the murine homolog of the human APC gene and develop spontaneous tumors throughout the intestine. Several observations implicate a role for altered DNA methylation in cancer pathogenesis: the global level of DNA methylation is generally lower but there is gene specific hypermethylation and DNA methyltransferase activity is usually higher in tumor cells than in normal cells. Global DNA methylation, gene specific DNA methylation, methyl metabolism and DNA methyltransferase activity will be evaluated in colon-derived human cells cultured in medium containing different chemical forms of selenium and different concentrations of folate, iron, or zinc and in animals fed diets containing different amounts of selenium, folate, iron or zinc. To determine the mechanisms for the chemopreventive effects of selenium and copper against colon cancer, gene specific macroarrays will be utilized and the effects of copper and selenium on signal transduction pathways for apoptosis and regulation of the cell cycle will be examined in cultured cells. The biological activity of zinc transcription factors will be studied using electrophoretic mobility shift assays or reporter gene constructs. Controlled human feeding studies and/or supplementation studies have and will be conducted to determine whether trace minerals shown to affect carcinogenesis in animal and cell culture models affect cancer susceptibility in humans. Humans will be fed different diets and fecal water will be analyzed for cytotoxicity, apoptosis, genotoxicity, free radical production and alkaline phosphatase activity. Lymphocytes will be analyzed for DNA methylation, expression of cancer related proteins and measures of oxidative stress/status. Serum from animals or humans fed different concentrations of trace minerals will be used in cell culture systems to investigate cancer susceptibility. Cancer is the second leading cause of death in the United States. It has been estimated that the cost for the treatment and care of this disease is approaching $200 billion per year. In addition to the economic impact, the development of cancer may prevent many from enjoying life to its fullest. It is believed that diet is the single greatest contributor to human cancer, possibly accounting for 30-40% of the disease. Dietary excesses, deficiencies and imbalances in trace mineral intake are factors that can affect cancer susceptibility. Thus, providing information about requirements and factors that affect those requirements of mineral elements should result in policies and programs that improve intakes of these nutrients that will result in a healthier population, decrease the burden of chronic disease, enhance the quality of life, and diminish health care expenditures. This research is related to National Program 107, Human Nutrition. The research addresses Performance Goal 3.1.1 of the National Program Action Plan: Human Nutrition Requirements. This research is relevant to Component 1: Nutrient Requirements because one of the priority objectives is to adapt current methods or develop new methods to identify specific disease preventing bioactive dietary factors and elucidate their mechanisms of action. Another priority objective is to use the biomarkers as screening tools to identify the specific bioactive factor(s) responsible for the effects. This research is also relevant to Component 2: Diet, Genetics, Lifestyle and the Prevention of Obesity and Disease. This research will identify the nutrient-relevant influences on gene expression that have consequences on human health and disease. Several trace minerals have been demonstrated to reduce the risk of developing several types of cancer, but the mechanism by which this occurs is unknown. These studies will address this problem, and thus are of interest to health professionals and policy makers. Understanding the mechanism by which trace elements inhibit cancer has the potential to impact recommendations of how much of the dietary trace mineral should be consumed daily; this in turn has the potential to impact how the medical establishment approaches cancer prevention and how the food industry prepares and/or fortifies specific foods. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2005) Initiate and optimize the HT 29 cell culture studies to determine whether cellular selenium status regulates the cyclin-dependent kinase pathway through the c-Myc gene (1.1). Initiate and optimize experiments to determine whether methylselenol causes colon cell cycle arrest and induces differential gene expression in the cyclin-dependent kinase pathway (1.3). Develop constructs and conduct experiments to determine whether thioredoxin reductase (TR) is regulated by selenium availability (dose and chemical form) as well as by ARE inducers and by oxidative stress (2.1 & 2.2). Determine whether phytochemicals in broccoli act synergistically with selenium in increasing TR activity (2.2). Complete the animal portion of the experiment to determine the effect of form and concentration of selenium on methionine sulfoxide activity and expression. Initiate metabolite and enzyme assays (2.6). Complete animal portion of experiment to study the interaction between selenium and folate. Initiate metabolite and enzyme assays. Begin restriction length genome scanning (RLGS) on select samples (3.3). Complete and report RLGS on initial (pilot) Ames dwarf mouse study (pre-3.2). Year 2 (FY 2006) Finish data collection on the Caco-2 cell culture studies designed to determine whether cellular selenium status regulates the cyclin-dependent kinase pathway and selenoenzyme (GPx) activities (1.1). Finish data collection on the experiments designed to determine whether methylselenol causes colon cell cycle arrest and induces differential gene expression in the cyclin-dependent kinase pathway (1.3). Perform bioinformatics search/study to understand gene data derived in the above cell culture experiments (1.1 &1.3). Prepare extracts from plants (other than broccoli) and conduct the studies to determine other compounds that transcriptionally upregulate the TR ARE independent of selenium (2.3). Complete analyses and report results of hypotheses 2.2-2.3. Complete analyses and report results from the experiment designed to determine the effect of form and concentration of selenium on methionine sulfoxide activity and expression (2.6). Complete enzyme and metabolite analysis (including RLGS) from experiment designed to study the interaction between selenium and folate (3.1). Initiate RLGS on Ames dwarf mice and age-matched wild type controls (3.2). Conduct developmental studies of tandem HPLC-ICP-MS analyses of plasma selenium (4.1). Plan selenium intervention trial (human) (4.2). Develop flow cytometric method for evaluating apoptosis in whole blood samples generated by selenium intervention study (4.2) Initiate human selenium intervention trial, including recruitment, conducting on-site briefings for perspective subjects, clinical screening of applicants, consented enrollment of volunteers, randomization to treatment, performance of baseline measurements and commencement of blinded intervention. (4.2) Year 3 (FY 2007) Optimize the colon cell culture conditions and start a study to determine how cellular selenium status regulates the mitogen-activated protein kinase pathway (1.2). Optimize the colon cell culture conditions and start a study to test the hypothesis that selenium-induced apoptotic signaling is different in normal versus transformed cells (1.4). Determine whether knockdown of TR will have minimal functional consequence for the cell because related ARE-regulated antioxidant systems will be compensatorily upregulated (2.4). Initiate preliminary experiments to test the hypothesis that simultaneous knockdown of TR and a second ARE-regulated protein (ferritin) will severely damage the ARE-regulated antioxidant network and result in severe cellular damage (2.5). Complete methylation assays and gene identification from RLGS in the selenium-folate and Ames dwarf mice studies (3.1 & 3.2). Initiate RLGS study in Ames mice fed various forms of selenium (3.3). Complete development of HPLC-ICP-MS method for selenium in plasma (4.1). Conduct selenium intervention trial, including the completion of subject enrollment, conduct interim subject visits and attendant analyses, and compilation of baseline data for all subjects (4.2). Year 4 (FY 2008) Finish cell culture experiments designed to determine how cellular selenium status regulates the mitogen-activated protein kinase pathway (1.2). Finish cell culture experiments designed to test the hypothesis that selenium-induced apoptotic signaling is different in normal versus transformed cells (1.4). Initiate studies to determine the role of differentially expressed genes, as discovered in previous cell culture studies, in mediating the anti-tumorigenic effect of selenium (1.5). Complete the experiments designed to determine the effect of simultaneous knockdown of TR and a second ARE-regulated protein (ferritin) (2.5). Complete the studies designed to determine whether knockdown of TR will have minimal functional consequence for the cell because related ARE-regulated antioxidant systems will be compensatorily upregulated (2.4). Complete analyses from the Ames dwarf mouse selenium study (3.3). Initiate Ames dwarf aberrant crypt study (contingency). Determine whether there is a relationship between methionine sulfoxide activity and/or expression and aberrant crypt formation in and aberrant crypt model (contingency for 2.6). Complete validation of HPLC-ICP-MS analytical procedure and report results (4.1). Complete selenium intervention trial; continue associated analytical work (4.2). Year 5 (FY 2009) Finish data collection and bioinformatics work on experiments designed to determine the role of differentially expressed genes, as discovered in previous cell culture studies, in mediating the anti-tumorigenic effect of selenium (1.5). If resources are available, initiate experiments to determine the effect of other dietary antioxidants (other than sulforaphanes and selenium) in ARE-protein knockdown cell models. Complete the Ames dwarf aberrant crypt study and related methionine sulfoxide studies. Complete analytical work for selenium intervention trial (4.1). Use HPLC-ICP-MS method to assess selenium status in samples from selenium intervention trial (4.2). 4a.List the single most significant research accomplishment during FY 2006. The elucidation of the mechanisms by which selenium regulates the cell cycle can lead to a better understanding of the nature of selenium’s essentiality and its role in disease prevention. Methylselenol has been hypothesized to be a critical selenium metabolite for anticancer activity. Methylselenol increased the protein levels of two antimestatic compounds TIMP-1 and TIMP-2) and inhibited the migration and invasion rate of fibrosarcoma cells in culture, decreasing the carcinogenic potential of activity of these tumor cells. Other cell culture work with human lymphocytes has shown that selenium deficiency inhibits cell cycle progression and decreases the mRNA expression of many cell cycle regulatory genes. Collectively, these results suggest that selenium is critical for human lymphocyte cell division, growth and prevention of cell death. Accomplishment aligns with Component 2: Relationship between diet, genetics and lifestyle and the risk for chronic disease. 4b.List other significant research accomplishment(s), if any. The methionine sulfoxide reductase (Msr) system (comprised of MsrA and MsrB) is important in repairing oxidized proteins. The system is responsible for reducing methionine sulfoxide [Met(O)] to methionine. MsrB is a selenoprotein and reduces the Met-R-(O) isomer whereas MsrA (a non-selenoprotein) reduces the Met-L-(O) isomer. Typically, the racemic Dabsyl-derivative Met-R/S-(O) is used to assay the enzyme activity. However, some researchers use the individual S or R derivatives to distinguish MsrA from MsrB. We developed a new assay that uses capillary electrophoresis for determination of MsrA and MsrB enzyme activities and of Dabsyl-derivative purity. We found typical preparations of Dabsyl-Met-R-(O) are contaminated with the S-isomer (10-25%). Furthermore, the activity towards Dabsyl-Met S-(O) is less than 10% of the activity towards Dabsyl-Met-R-(O). Taken together, our data suggest that the selenoprotein MsrB accounts for the greatest percentage of enzyme activity in the Msr system and that researchers who use the racemic mixture Dabsyl-R/S-Met(O) to measure Msr activity need to realize that most of the associated activity is accounted for by the selenoprotein MsrB. This is important because most researchers ignore this fact and, therefore, results that are attributable to selenium have not been realized. Accomplishment aligns with Component 2: Relationship between diet, genetics and lifestyle and the risk for chronic disease. The LoDoSe trial (4.2), which was planned and initiated in FY2006, constitutes the first human trial in which selenium doses less than 200 mcg/day have been used in non-deficient subjects. This is important for the reason that, while the 200 mcg/day dose has been found to reduce cancer risks in healthy Americans, further analyses suggested that selenium doses much less than that may be effective. That prospect has direct implications to the US food industry which can produce foods capable of providing 50-100 mcg selenium per day through geographic sourcing, or selenium-fortification. The results of the LoDoSe trial will provide the evidence base for the development of foods and food policy for reducing cancer risks by achieving target plasma selenium levels. Accomplishment aligns with Component 1: Nutrient requirements. 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. This research is directly related to the ARS National Human Nutrition Research Plan 107, Component 2: Diet, Genetics, Lifestyle and the Prevention of Disease, and directly contributes to the accomplishment of ARS Strategic Plan Goal #4, Improve the Nation’s Nutrition and Health. Butyrate treatment inhibits the migration and invasion potential of tumor cells: Butyrate, a product of the bacterial fermentation of dietary fiber, has been hypothesized to be directly related to the lower risk of colon cancer. It is important to test and to characterize the invasive ability of tumor cells as affected by long exposure to low concentrations of butyrate. Our results demonstrate that prolonged and low-dose butyrate treatment (0.5 mmol/L butyrate, similar to a moderate fiber diet) inhibits pro-MMP-2 activation and tumor cell migration/invasion potential. Our data provide a possible mechanism for butyrate’s anticarcinogenic properties. These data support the health claim of a high-fiber diet and provide a mechanism to link fiber and decreased cancer risk. Thioredoxin reductase is regulated by sulforaphane: Thioredoxin reductase, a selenium-requiring protein, is transcriptionally regulated by sulforaphane from broccoli. Broccoli contains many other compounds that may inhibit cancer, and some of this inhibition may be mediated by turning on ARE containing enzymes such thioredoxin reductase. This hypothesis was investigated by adding compounds known to be in broccoli to cells in culture containing a reporter gene construct of thioredoxin reductase. Although ascorbic acid resulted in modest induction, the results convincingly demonstrated that sulforaphane was responsible for almost all of the ARE-mediated activation of thioredoxin reductase. This research provides a plausible mechanism to explain how broccoli consumption may inhibit cancer. Furthermore, this study provides a tool that can be used to screen various compounds to determine their cancer-fighting ability. Glycine N-methyltransferase, a tumor susceptibility gene, is decreased in selenium deficiency: Determined that the activity of the enzyme glycine N-methyltransferase (GNMT) is decreased by selenium deprivation. GNMT, which has been shown to be a tumor susceptibility gene, is a regulator of tissue S-adenosylmethionine concentration, and in liver, is a major folate binding protein. Thus, GNMT may induce changes in tissue folate status resulting in chromosome breakage or abnormal DNA methylation. Second, GNMT is an enzyme participating in detoxification. In addition, GNMT may have a protective effect against the exposure to carcinogens by decreasing DNA adduct formation. Thus, the decrease in this enzyme may explain some of the effects of selenium deficiency. This study affirms the importance of the interaction between dietary selenium and folic acid and suggests that alterations in selenium status may affect folate status and vice versa. This may prove most important in the nutrition of those humans who may have low folate and low selenium status. That is, supplementation of one without the other may be more detrimental than beneficial. Transsulfuration is markedly enhanced in the long-lived Ames dwarf mouse: Shown by using tracer studies that transsulfuration is markedly enhanced in the Ames dwarf mouse. The Ames dwarf mouse lives substantially longer and has a lower incidence of cancer than the wild type. This tracer study, along with real-time RT PCR findings of genes associated with methionine metabolism, provides a plausible mechanism for the increased glutathione found in the dwarf mice. This supports the hypothesis that these mice have enhanced oxidative defense capabilities. This study gives insight to what mechanisms in animals are important in aging and in decreasing the risk of cancer; similar mechanisms (and how nutrition affects them) could then be studied in humans. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Information on how trace elements (specifically selenium) affect mechanisms related to carcinogenesis was presented at numerous workshops and scientific meetings. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Eight presentations/publications were made: three Grand Forks Herald articles, “B12: It’s more Important than You Think,” “Diet, Lifestyle Play Role in Preventing Cancer,” and “Diet High in Folic Acid Can Help Fight Disease;” and talks at the East Grand Forks Senior Citizen’s Center entitled “Food for Thought” and “What Is Selenium?;” a seminar at South Dakota State University “The Role of Selenium in Reducing Cancer Risk;” a talk at the Grand Forks Rotary Club “The Role of the USDA in Researching Questions of Diet and Health;” and a lecture at the 9th International Symposium on Metal Ions in Biology and Medicine, Lisbon, “Determining Healthful Intakes of Selenium.” Review Publications Stranges, S., Marshall, J.R., Trevisan, M., Natarajan, R., Combs, G.F., Farinaro, E., Reid, M.E. 2006. Effects of selenium supplementation on cardiovascular disease incidence and mortality: secondary analysis in a randomized clinical trial. American Journal of Epidemiology. 163:694-699. Uthus, E.O., Ross, S., Davis, C.D. 2006. Differential effects of dietary selenium (se) and folate on methyl metabolism in liver and colon of rats. Biological Trace Element Research. 109:201-214. Zeng, H., Briske-Anderson, M.J., Idso, J.P., Hunt, C. 2006. The selenium metabolite methylselenol inhibits the migration and invasion potential of HT1080 tumor cells. Journal of Nutrition. 136:1528-1532. Chang, W.P., Combs, G.F., Scanes, C.G., Marsh, J.A. 2005. The effects of dietary vitamin E and selenium deficiencies on plasma thyroid and thymic hormone concentrations in the chicken. Developmental and Comparative Immunology 29:265-273. Combs, Jr., G.F. 2005. Importance of selenium in human nutrition. Agrifood Research Reports 69. Proceedings, Twenty Years of Selenium Fertilization. p. 60-70. Brown-Borg, H.M., Rakoczy, S.G., Uthus, E.O. 2004. Growth hormone alters components of the glutathione metabolic pathway in Ames dwarf mice. Annals of the New York Academy of Sciences. 1019:317-320. Patterson, B., Wasteny, M., Combs, G.F., Brindak, M., Patterson, K.K., Veillon, C., Taylor, P., Levander, O.A. 2006. Selenium metabolism in humans: Response of kinetic pools in plasma to 2 yr supplementation. The FASEB Journal Book of Abstracts, Volume 20, No. 5, p. A1069:673.11. Uthus, E.O., Ross, S. 2006. Dietary selenium (Se) but not folic acid (FA) affects the activity and message of rat liver betaine homocysteine methyltransferase (BHMT)[abstract]. Journal of Federation of American Societies for Experimental Biology. 20(4):A429. Zeng, H., Briske Anderson, M.J., Idso, J.P., Hunt, C. 2006. Selenium metabolite methylselenol inhibits migration and invasion potential of HT1080 tumor cells [abstract]. FASEB J. 20(5):A1011. Combs, Jr., G.F. 2006. Drinking water as a source of mineral nutrition. In: Institute of Medicine of the National Academies, editors. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC:National Academies Press. p. 295-304. Uthus, E.O., Brown-Borg, H.M. 2006. Methionine flux to transsulfuration is enhanced in the long living Ames dwarf mouse. Mechanisms of Aging and Development. 127:444-450.