2005 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? What 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, fruit 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 the milestones (indicators of progress) from your Project Plan. 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 HT 29 cell culture studies designed to determine whether cellular selenium status regulates the cyclin-dependent kinase pathway through the c-Myc gene (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). 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 compensatorly 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). 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 compensatorly 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). 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. 4a.What was the single most significant accomplishment this past year? 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 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. IMPACT: These data support the health claim of a high-fiber diet and provide a mechanism to link fiber and decreased cancer risk. 4b.List other significant accomplishments, if any. 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. IMPACT: 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. IMPACT: 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: Showed 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 that 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. IMPACT: 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. 4c.List any significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments over the life of the project, including 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 accomplishmnet of ARS Strategic Plan Goal #4, Improve the Nation's Nutrition and Health. [This report is the first for this CRIS project (certified in 2005), thus the accomplishments for the life of the project are incorporated in sections 4a and 4b.] 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). Two presentations were made in the fourth quarter FY04: A Grand Forks Herald article entitled “Plant-based diet may be healthiest choice” and a talk to the Asian Horticultural Society entitled “Efficacy of plant-based compounds for cancer reduction.” Review Publications Brown-Borg, H.M., Radoczy, S.G., Uthus, E.O. 2004. Growth hormone alters methionine and glutathione metabolism in Ames dwarf mice. Mechanisms of Ageing and Development 126:389-398. Davis, C.D., Uthus, E.O. 2004. DNA methylation, cancer susceptibility, and nutrient interactions. Experimental Biology and Medicine. 229:988-995. Uthus, E.O., Davis, C.D. 2005. Dietary arsenic affects dimethylhydrazine-induced aberrant crypt formation and hepatic global DNA methylation and DNA methyltransferase activity in rats. Biological Trace Element Research. 103:133-145. Zeng, H., Briske Anderson, M.J. 2005. Prolonged butyrate treatment inhibits the migration and invasion of potential HT1080 tumor cells. Journal of Nutrition. 135:291-295. Zeng, H., Uthus, E.O., Combs, G.F. 2005. Mechanistic aspects of the interaction between selenium and arsenic. Inorganic Biochemistry. 99:1269-74. Combs, G.F. 2005. Current evidence and research needs to support a health claim for selenium and cancer prevention. Journal of Nutrition. 135:343-347. Combs, Jr., G.F. 2004. Status of Selenium in Prostate Cancer Prevention. British Journal of Cancer. 91:195-199. Combs Jr., G.F., Patterson, B., Brindak, M., Midthune, D., Taylor, P., Veillon, C., Patterson, K., Hill, D., Levander, O.A. 2005. Thyroid hormone levels in subjects supplemented with oral selenomethionine [abstract]. The Federation of American Societies for Experimental Biology Journal. 19(5):A1016. Patterson, B., Wastney, M.E., Combs Jr., G.F., Brindak, M., Patterson, K.Y., Veillon, C., Taylor, P., Levander, O.A. 2005. Se metabolism in humans is altered by long-term supplementation [abstract]. The Federation of American Societies for Experimental Biology Journal. 19(5):A1015. Uthus, E.O., Ross, S., Davis, C. 2005. Differential effects of dietary selenium (Se) and folate on methyl metabolism in liver and colon or rats [abstract]. The Federation of American Societies for Experimental Biology Journal. 19(5):A1013. Zeng, H., Briske-Anderson, M. 2005. Inhibitory effect of prolonged-butyrate treatment on migration and invasion of HT1080 tumor cells [abstract]. The Federation of American Societies for Experimental Biology Journal. 19(5):A1693.