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? This CRIS, which includes the Vitamins and Carcinogenesis Laboratory and the Nutrition and Cancer Biology Laboratory, is designed to further our understanding of how dietary habits, nutritional supplementation, and other nutritional interventions can be used to prevent cancer. New knowledge attained by the efforts of this CRIS can then be translated into public health initiatives that effectively reduce the burden of cancer in our society. To accomplish this goal, we examine the complex roles that diet plays in modifying metabolic and genetic pathways that lead to human carcinogenesis. Our program is presently focused on the carotenoids, retinoids, and nutrients involved in one-carbon metabolism (methionine, choline and the B-vitamins, folate, B12, B6, and B2). These nutrients have drawn considerable attention with regard to their potential for human cancer prevention. This work is designed to determine how these dietary compounds interact with genetic background to modify molecular and signaling pathways that alter the development of common cancers, such as those of the lung, stomach, liver and colorectum. This research also examines how this response is modified by other exogenous factors (e.g., tobacco smoking, alcohol and chemical carcinogen exposures). Several different approaches, encompassing work in animals, cell culture, and humans, are used in this effort. Cancer continues to be the second most common cause of mortality in the United States (US) as well as in most developed countries. As the incidence of cardiovascular disease has declined, the significance of the burden that cancer puts on our society has become even more important. Most cancers can be considered as a ‘degenerative disease of aging’, since elder age is a major risk factor for cancer. The efforts of this CRIS are highly relevant to the elders in our society. However, cancers appear to develop over many years in an environment that promotes their evolution, and therefore dietary interventions designed to prevent cancer are relevant to younger populations as well since it is the dietary habits of young and middle adulthood that appear to determine the likelihood of cancer developing later on. The efforts of this CRIS are highly aligned with two components of the National Program 107 – Human Nutrition. Since a major mission of this CRIS is to define the relationship between particular nutrients, genetic and environmental factors and the risk of cancer, our studies are related to components: 7 - Health promoting properties of plant and animal foods and 6 - Prevention of obesity and disease: Relationship between diet, genetics and lifestyle. 2.List the milestones (indicators of progress) from your Project Plan. Vitamins and Carcinogenesis: Objective 1: To determine the complex roles that ‘one-carbon nutrients’ (methionine, choline and the B-vitamins, folate, B12, B6, and B2), as well as components of the diet that are one-carbon antagonists (such as alcohol) play in modifying metabolic and genetic pathways that lead to human cancer. Objective 2: To define how the mechanistic knowledge acquired through objective 1 should be used to modify dietary habits, nutritional supplementation, and other nutritional interventions in order to prevent cancer. New advances made by this laboratory can thus be translated into public health initiatives that effectively reduce the burden of cancer in our society. Milestones: 2005 1. Conduct a study in mice to determine the effect of folate depletion, and folate depletion combined with deficiencies of vitamins B2, B6, and B12, on colonic 1-carbon metabolites, on DNA stability, and on histopathologic indicators of cancer (animal study 1). Objective 1 2. Initiate the development of a genetically engineered mouse that possesses the murine homologue of the common 677 homozygous MTHFR polymorphism in humans. Objective 1 3. Initiate two human intervention trials designed to assess the biochemical and molecular changes in the colon due to folate depletion and folate supplementation. Objectives 1 and 2 4. Determine the effects of different levels of folate availability on the stability of the mitochondrial genome in rat colon and in cultured human colon cells. Objective 1 2006 1. Analyze the blood and tissue samples from animal study 1 (see milestone 1, 2005), and analyze the data so obtained. Objective 1 2. Complete development of the 677 MTHFR mouse, and conduct experiments to demonstrate that it behaves physiologically similar to the human. Objective 1 3. Initiate studies in a strain of mice genetically-predisposed to colon cancer (apc1638N) to determine the susceptibility of this strain to modulation of carcinogenesis by folate status (animal studies 2a and 2b). Objective 1 4. Analyze tissue and blood samples from the human supplementation and depletion trials, and continue to enroll subjects. Objectives 1 and 2 2007 1. Complete animal studies 2a and 2b (see above) to determine the modulation of colon carcinogenesis in apc1638N animals by folate status. Objective 1 2. Perform a mouse study to determine how the superimposition of vitamins B2, B6, and B12 depletion further modulates colonic carcinogenesis in the APC1638N mouse (animal study 3). Objective 1 3. Complete recruitment of human subjects for supplementation and depletion trials, analyze samples and data. Objectives 1 and 2 2008 1. Complete the conduct of animal experiment 3 (see 2007 milestones) and perform data analysis. Objective 1 2. Begin animal study 4 with the MTHFR 677 mouse (see milestones, 2006) to determine how this human polymorphism alters biochemical and molecular endpoints in the mouse colon that are relevant to carcinogenesis. Objective 1 2009 1. Complete animal study 4 and analyze data. Objective 1 2. Determine how the MTHFR polymorphism alters the risk of colon cancer by using a mouse generated by crossing the C677 MTHFR mouse with the APC1638N mouse (animal study 5). Objective 1 Nutrition and Cancer Biology Laboratory: The goal of the Nutrition and Cancer Biology Laboratory is to understand the mechanistic basis for the possible chemopreventive efficacy of lycopene against lung cancer development and the metabolic pathway of lycopene under well-controlled experimental conditions, using the ferret model, which is highly analogous to humans. Objective 1 To establish the dose response effects of lycopene in plasma and lung tissue with and without smoke exposure. Objective 2 To establish the potential for lycopene inhibition of smoke-induced lung lesions and mechanism(s) of action. Objective 3 To characterize both the expression and activity of carotene 9’, 10’-monooxygenase (CMO, an excentric cleavage enzyme for carotenoids) in the metabolism of lycopene with and without smoke exposure, and the biological effect of the metabolite(s) of lycopene. Milestones: 2005 1. To establish the ferret as a smoke-related lung cancer model by inducing lung preneoplastic lesions and tumors in the carcinogen treated ferrets, which mimics humans for investigating dose-response effect of lycopene. Objectives 1 and 2 2. Clone both IGF-1 and IGFBP-3 genes from the ferret and examine the expression of IGF-1/IGFBP-3 using the real-time Polymerase Chain Reaction (PCR) method, which provides a molecular tool for investigating the mechanism for the chemopreventive efficacy of lycopene. Objective 2 3. Clone carotene 9’, 10’-monooxygenase (CMO) gene from the ferret, express the CMO gene using the baculovirus system, examine the CMO activity and produce an antibody against CMO, which provides a molecular basis for investigating the metabolic pathway of lycopene. Objective 3 2006 1. Determine the effectiveness of lycopene either in low (equivalent to 15 mg/day in human) and in moderate (equivalent to 30 mg/day in humans) doses on plasma and tissue levels of lycopene and development of lung preneoplastic lesions and tumors in the carcinogen treated, cigarette smoke-exposed ferret model. Objective 1 and 2 2. Determine the effectiveness of lycopene either in low and in moderate doses on smoke-induced oxidative stress and DNA damage in the carcinogen treated, cigarette smoke-exposed ferret model. Objectives 1 and 2 3. Examine both the expression and activity of carotene 9’, 10’-monooxygenase and the formation of apo-10’-lycopenoids from lycopene in ferrets supplemented with either low or moderate doses, with or without smoke exposure. Objective 3 2007 1. Determine the effectiveness of lycopene in high (equivalent to 90 mg/day in humans) dose on plasma and tissue levels of lycopene and the development of lung preneoplastic lesions and tumors in the carcinogen-treated, cigarette smoke-exposed ferret model. Objectives 1 and 2 2. Determine the effectiveness of high dose lycopene supplementation on smoke-induced oxidative stress and DNA damage in the carcinogen-treated, cigarette smoke-exposed ferret model. Objectives 1 and 3 3. Determine both the expression and activity of carotene 9’, 10’-monooxygenase and the formation of apo-10’-lycopenoids from lycopene in ferrets supplemented with high doses of lycopene, with or without smoke exposure. Objective 3 2008 1. Determine if lycopene inhibits smoke-induced lesions by up-regulating insulin-like growth factor binding protein-3 (IGFBP-3) as a molecular target. Objective 2 2. Determine if lycopene interrupts the signal transduction pathway of insulin-like growth factor-1 (IGF-1) as a mechanism for the chemopreventive efficacy of lycopene. Objective 2 3. Determine the possible mechanism(s) of induction of the carotene 9’, 10’-monooxygenase by lycopene supplementation in ferrets with or without smoke exposure. Objective 3 2009 1. Determine the biological activity of lycopene metabolites, apo-10’-lycopenoids, against lung preneoplastic lesions and tumors in the carcinogen treated, cigarette smoke-exposed ferret model. Objective 3 2. Determine if lycopene metabolites, apo-10’-lycopenoids, inhibits smoke-induced lesions by up-regulating insulin-like growth factor binding protein-3 (IGFBP-3) as a molecular target. Objective 3 3. Determine if lycopene metabolites, apo-10’-lycopenoids, interrupts the signal transduction pathway of insulin-like growth factor-1 (IGF-1) as a mechanism for the chemopreventive efficacy of lycopene. Objective 3 4a.What was the single most significant accomplishment this past year? Oral Cancer and One-carbon Nutrients During the last CRIS cycle the Vitamins and Carcinogenesis Laboratory collected samples from 35 smokers and 21 non-smokers in a cross-sectional study to determine whether depletion of one-carbon nutrients in the mouth contribute to the high incidence of oral cancer in smokers. We completed the data analysis this year and found that smoking is associated with a ~50% drop in total folate concentration in the oral mucosa that cannot be explained by differences in dietary intake, and that these quantitative changes are accompanied by qualitative changes in the types of folates present in the mouth. In a small intervention trial that followed the cross-sectional study, we demonstrated that oral supplementation with 1000 micrograms of folate per day over one month restored oral folate levels to those observed in non-smokers, and altered genomic DNA methylation but did not alter uracil incorporation or micronuclei in the buccal cells. 4b.List other significant accomplishments, if any. Small Defects in the Colonic Lining are Highly Related to Age and Cigarette Smoking The Vitamin and Carcinogenesis Laboratory completed data analysis of a study of approximately 80 men undergoing colonoscopy that was conducted during the previous CRIS cycle. Scientists demonstrated that the prevalence of small defects in the colonic lining, which are presumed to be the earliest observable precursor of cancer (aberrant crypt foci) is highly related to both the age of the individual as well as the extent of cigarette smoking in that individual. Ferrets and Lung Cancer Chemoprevention Previously, the Nutrition and Cancer Biology Laboratory demonstrated that the ferret (Mustela putorious furo) offers a model for use in mimicking the conditions of carotenoid (e.g., beta-carotene and lycopene) intervention studies in smokers, due to the similarities between ferret and human in terms of carotenoid absorption, metabolism, tissue distribution and levels and biological functions. However, the susceptibility of smoke/carcinogen-exposed ferrets toward actual tumor production in the lungs is unknown, which limits its value for both research in lycopene function and lung cancer prevention. This year, we performed a six-month in vivo study in ferrets exposed to both tobacco smoke and a carcinogen [4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK] found in cigarette smoke. Six 6 of 12 ferrets exposed to both carcinogen and cigarette smoke developed grossly identifiable lung tumors, whereas none of 9 ferrets from the sham treatment group developed lung lesions. The histopathological types of these tumors (squamous cell carcinoma, adenosquamous carcinoma and adenocarcinoma) in ferret lungs are very similar to those in humans. In addition, 10 of 12 ferrets exposed to both carcinogen and cigarette smoke developed preneoplastic lesions (squamous metaplasia, dysplasia, and atypical adenomatous hyperplasia) with complex growth patterns, whereas the sham groups did not show any of these lesions. This study, for the first time, established the ferret as lung cancer model, providing an excellent and unique model for studying lung cancer chemoprevention with dietary agents such as lycopene, and for studying the molecular mechanisms of carcinogenesis in the earlier stages of smoke-related lung cancer. Effects of ß-carotene Supplementation is Dose Related The Nutrition and Cancer Biology Laboratory has previously demonstrated that low-dose beta-carotene can prevent the decreased lung retinoic acid and the smoke-induced lung lesions. This year we investigated whether smoke exposure and/or ß-carotene supplementation could affect Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and p53 in the lungs of ferrets. Ferrets were subjected to cigarette smoke exposure and either a high- or low-dose of beta-carotene for 6 months. There were greater protein levels of phosphorylated JNK, p38, and c-Jun, but lower levels of MAPK phophatase-1 (MKP-1) in groups exposed to smoke and/or high dose ß-carotene. Both phosphorylated-p53 and total p53 were substantially increased in the lungs of these groups. In contrast, low dose ß-carotene greatly attenuated the smoke-induced phosphorylation of JNK, p38, c-Jun, p53, and total p53, accompanied by up-regulated MKP-1. Smoke exposure increased MAPK kinase-4 (MKK4) phosphorylation regardless of ß-carotene supplementation. These data indicate that restoration of retinoic acid and MKP-1 by low dose ß-carotene in the lungs of ferrets may prevent the smoke-induced activation of the JNK-dependent signaling pathway, p38 MAPK, and the associated phosphorylation of p53, thereby lowering the risk of the smoke-related lung lesions. These data provide supportive evidence that the beneficial versus detrimental effects of ß-carotene supplementation are related to the dosage of ß-carotene administered. Alcohol-impaired Hepatic Vitamin A Status Contributes to the Decreased Plasma IGF-I Level and Hepatic IGF-I Expression in Alcoholics Chronic and excessive alcohol intake is associated with a number of impairments in biochemical metabolism and nutritional status, such as vitamin A. Chronic ethanol intake also decreases levels of insulin-like growth factor-I (IGF-I), which plays a critical role in the regulation of cellular proliferation, differentiation and apoptosis in wide variety of cells and tissues. The Nutrition and Cancer Biology Laboratory examined both plasma IGF-I level and hepatic IGF-I mRNA expression in alcohol-fed rats with or without vitamin A acid (the most active form of vitamin A) supplementation for six months. We observed that hepatic IGF-I mRNA levels and plasma IGF-I concentration were decreased significantly in alcohol-fed rats, as compared to controls. In contrast, vitamin A acid supplementation in ethanol fed rats partially restored both hepatic IGF-I mRNA levels and plasma IGF-I concentration, as compared with rats fed ethanol alone. These data suggest that alcohol-impaired hepatic vitamin A status contributes to decreased plasma IGF-I level and hepatic IGF-I expression in alcoholics. 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 CRIS was created in the latter half of FY 04. The significant accomplishments to date include the work of the Vitamins and Carcinogenesis Laboratory on oral cancer and one-carbon nutrients in determining whether depletion of one-carbon nutrients in the mouth contribute to the high incidence of oral cancer in smokers. In a cross-sectional study of 35 smokers versus 21 non-smokers, we found that smoking is associated with a ~50% drop in total folate concentration in the oral mucosa that cannot be explained by differences in dietary intake, and that these quantitative changes are accompanied by qualitative changes in the types of folates present in the mouth. In a small intervention trial that followed the cross-sectional study, we demonstrated that oral supplementation with 1000 micrograms of folate per day over one month restored oral folate levels to those observed in non-smokers, and altered genomic DNA methylation but did not alter uracil incorporation or micronuclei in the buccal cells. This study lays important foundation work for subsequent studies that will explore whether folate supplementation can prevent oral cancer. This work is related to National Program 107 – Human Nutrition: 6 - Prevention of obesity and disease: Relationship between diet, genetics and lifestyle and to Performance measure 4.1.2: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods Research into dietary chemoprevention against lung carcinogenesis has been limited by the lack of appropriate animal models that closely mimic smoking-related human lung cancer. Ferrets (Mustela putorius furo) have been used to study the biologic activities of carotenoids against smoke-induced lung lesions, however this model has yet to be thoroughly established and validated. The Nutrition and Cancer Biology Laboratory has demonstrated that the ferret is an excellent model for human lung cancer resulting from tobacco smoke exposure and can be used for carotenoid supplementation and lung cancer chemoprevention interventions as well as in mechanistic studies that attempt to describe molecular changes relevant to human lung cancer and modification by dietary factors. This research is related to objectives 1 and 2, milestone 1: To establish ferret as lung carcinogenesis model by inducing lung preneoplastic lesions and tumors in the carcinogen-treated, cigarette smoke-exposed ferrets and to the National Program 107 component 6: Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle, and to Performance Measure 4.1.2: Improve Human health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 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? Research findings from this CRIS has been published in peer-reviewed journals and presented to scientists at professional 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). Presentations Mason JB. January 24, 2005: Dr. Mason presented a talk at the National Press Club in Washington D.C. about the potential cancer preventive properties of folate. Wang XD. Invited speaker on “Can smoke-exposed ferrets be utilized to unravel the mechanisms of action of lycopene?” in the NCI/NIH workshop "Promises and Perils of lycopene/tomato supplementation and cancer prevention”, Bethesda, Maryland, February, 2005 Wang XD. Invited speaker on "Carotenoids, gene regulation and cancer prevention”, 2005 Experimental Biology meeting, April, San Diego, CA, 2005 Review Publications Liu, C., Russell, R.M., Wang, X. 2004. Low dose beta-carotene supplementation of ferrets attenuates smoke-induced lung phosphorylation of JNK, p38 MAPK , and p53 proteins. Journal of Nutrition. 134(10):2705-2710. Lian, F., Chung, J., Russell, R.M., Wang, X. 2004. Alcohol-reduced plasma igf-i levels and hepatic igf-i expression can be partially restored by retinoic acid supplementation in rats. Journal of Nutrition. 134(11):2953-2956. Dan, Z., Popov, Y., Patsenker, E., Preimel, D., Liu, C., Wang, X., Seitz, H.K., Schuppan, D., Stickel, F. 2005. Hepatotoxicity of alcohol-induced polar retinoid metabolites involves apoptosis via loss of mitochondrial membrane potential. Journal of Federation of American Societies for Experimental Biology. [FJ Express 10.1096/fj.04-2809fje, published online February 24, 2005] Available at http://www.fasebj.org. Kim, Y., Liu, X.S., Liu, C., Smith, D.E., Russell, R.M., Wang, X. 2005. Induction of pulmonary neoplasia in the smoke-exposed ferret by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nnk): a model for human lung cancer. Cancer Letters. (Epub) Available at http://sciencedirect.com. doi:10.1016/j.canlet.2005.03.052 Wang, X. 2005. Biological activity of lycopene against smoke-induced lung lesions: targeting igf-i/igfbp-3 signal transduction pathway. In: Packer, L., Obermueller-Jevic, U., Kraemer, K., Sies, H. Editors. Champaign, Illinois, USA. Carotenoids and Retinoids: Molecular Aspects and Health Issues. AOCS Press, p. 168-181. Lian, F., Russell, R., Wang, X. 2005. Apo-10'-lycopenoic acid, a metabolite of lycopene, inhibits the proliferation of non-small cell lung cancer cells [abstract]. Journal of Federation of American Societies for Experimental Biology. 19:A1458. Kim, Y., Russell, R., Wang, X. 2005. Combined antioxidant (beta-carotene, alpha-tocopherol and ascorbic acid) supplementation protects against oxidative DNA damage and chemical carcinogen (NNK) induced lung carcinogenesis in smoke exposed ferrets [abstract]. Journal of Federation of American Societies for Experimental Biology. 19:A474. Mernitz, H., Smith, D., Zhu, A.X., Wang, X. 2005. Effects of 9-cis retinoic acid supplementation on cox-2 and rar-beta levels and tumor formation in the lungs of nnk-induced a/j mice [abstract]. Journal of Federation of American Societies for Experimental Biology. 19:A78. Chavez, P.R., Russell, R., Mason, J.B., Ordovas, J.M., Wang, X. 2005. Effects of chronic ethanol feeding on hepatic cell proliferation, ERK activation and altered hepatic foci formation in diethylnitrosamine-initiated rats [abstract]. Journal of Federation of American Societies for Experimental Biology. 19:A78. Crott, J.W., Choi, S., Branda, R.F., Mason, J.B., 2005. Accumulation of mitochondrial DNA deletions is age, tissue and folate-dependent in rats. Mutation Research. 579(1):63-70. Friso, S., Girelli, D., Trabetti, E., Olivieri, O., Guarini, P., Pignatti, P., Corrocher, R., Choi, S. 2005. The MTHFR 1298A>C polymorphism and genomic DNA methylation in human lymphocytes. Cancer Epidemiology Biomarkers and Prevention. 14(4):938-943. Jang, H., Keyes, M.K., Dolnikowski, G., Liu, Z., Crott, J.W., Mason, J.B., Choi, S. 2005. Folate supplementation increases genomic DNA methylation of the lung in young, but not in old mice [abstract]. Journal of Federation of American Societies for Experimental Biology. Paper No. 161.4. Moxon, D., Raza, M., Kenney, R., Ewing, R., Arozullah, A., Mason, J., Carroll, R.E. 2005. Relationship of aging and tobacco use with the development of aberrant crypt foci in a predominately African-American population. Clinical Gastroenterology and Hepatology. 3(3):271-8. Choi, S., Friso, S., Keyes, M.K., Mason, J.B. 2005. Folate supplementation increases the genomic dna methylation in the liver of elder rats. British Journal of Nutrition. 93(1):31-5. Mason, J. 2005. Nutrition and gastroenterology: A mutually supportive partnership. Nutrition in Clinical Care. 7(3):91.