Progress 10/01/08 to 09/30/13
Outputs
OUTPUTS: This project had two research components: Project A hypothesized that dietary sources of nitrate and nitrite may be associated with the risk of colorectal cancer progression. Previous data and research work provided additional support for that hypothesis. The work found that dietary patterns high in nitrate and nitrite from fruits and vegetables may be responsible for part of the hypotensive effect of the Dietary Approaches to Stop Hypertension diet. Results were extended by applying nitrite to colon carcinoma cells at different stages of transformation to ascertain potential preventive or promotional risks. Studies were also implemented to determine if sodium nitrite in drinking water can affect colitis severity in a murine model of colitis called the SMAD3 knock out mouse. The preliminary data indicated that low (50 mg/liter) and high (2 grams/liter) concentrations of sodium nitrite dose-dependently decrease colitis scores compared to non-nitrite fed control animals. Project B hypothesized that an energy restricted ketogenic diet affects the growth of gioblastoma in humans. It was titled "Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 (OXCT1) and Glial Fibrillary Acidic Protein (GFAP) Content in Human Astrocytoma and Gliosarcoma: Implication for Ketone Utilization in Cancer" Results from project B demonstrated that astrocytomas and gliosarcomas express low levels of OXCT1 and BDH1 in human GBM tumors while BDH2 and ACAT1 are expressed in these tumors. This limited data supported the hypothesis that astrocytomas may express low leve3ls of the enzymes necessary to utilize ketones for energy and growth. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Project A : Overall, the data indicates that nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at high concentrations in cells representing stage 4 colon carcinomas. Project B: Preliminary results from human GBM indicates that an energy restricted ketogenic diet is a biologically plausible therapy for GBM.
Publications
- Jiang H, Tang Y, Garg HK, Parthasarathy DK, Torregrossa AC, Hord NG, Bryan NS. 2012. Concentration- and stage-specific effects of nitrite on colon cancer cell lines. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society. 26(4):267-73.
- Hord NG. 2011. Dietary nitrates, nitrites, and cardiovascular disease.. Current atherosclerosis reports. 13(6):484-92.
- Hord NG, Ghannam JS, Garg HK, Berens PD, Bryan NS. 2011. Nitrate and nitrite content of human, formula, bovine, and soy milks: implications for dietary nitrite and nitrate recommendations. Breastfeeding medicine : the official journal of the Academy of Breastfeeding Medicine. 6(6):393-9.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Project A findings provide additional support for our hypothesis that dietary sources of nitrate and nitrite may be associated with health benefits. Our published results and work described below support our hypothesis that nitrite may be associated with risk of colorectal cancer progression dependent upon stage of colon carcinoma. There is growing evidence revealing that nitrate and nitrite metabolism occurs in blood and tissue to form NO and other bioactive nitrogen oxides, representing an alternative to classical L-arginine-NO synthase -NO signaling pathway. NO is involved in neurotransmission, vasodilation, inflammation, and immunity and is also believed to play roles in multiple stages of various cancers. Our collaborative group has reported that dietary patterns high in nitrate and nitrite from vegetables and fruits may be responsible for part of the hypotensive effect of the Dietary Approaches to Stop Hypertension diet. These data have been presented at invited seminars at the University of Arizona's Nutrition and Health meeting as well as local and state dietetic association meetings. This year we have extended our results by applying nitrite to colon carcinoma cells at different stages of transformation to ascertain potential preventive or promotional risks. This work is in press for the journal Nitric Oxide. We have also implemented studies to determine if sodium nitrite in drinking water can affect colitis severity in a murine model of colitis called the SMAD3 knock out mouse. This model initiates colitis with a bacterium called H. hepaticus and colitis development is measured using colitis score by Dr. Inge Langhor, our collaborating pathologist. Our preliminary data indicate that low (50 mg/liter) and high (2 grams/liter) concentrations of sodium nitrite dose-dependently decrease colitis score compared to non-nitrite fed control animals. Project B was developed using funds from the American Cancer Society, in collaboration with Drs. L. Karl Olson and Howard Chang, to determine if ketone metabolizing enzymes in brain cancer tissues are expressed, as hypothesized, at low levels. This work, titled "Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 (OXCT1) and Glial Fibrillary Acidic Protein (GFAP) Content in Human Astrocytoma and Gliosarcoma: Implications for Ketone Utilization in Cancer" indicate that the expression of OXCT1 and BDH1 is negative in human GBM tumors while BDH2 and ACAT1 are expressed in these tumors. Quantitation and publication of these data will occur upon completion of data analysis and manuscript preparation. The human study protocol to implement the energy restricted ketogenic diet for GBM patients has received approved from appropriate IRB committees at MSU and has received funding for patient accrual. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Scientists and Registered Dietitians who inform patient food choices for chronic disease risk will benefit from these data. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Project A: Consumption of specific food components is associated with risk of colorectal cancer (CRC). Nitrates and nitrites, used exogenously in processed meats to enhance taste and microbial food safety and associated with increased cancer risk, are also endogenous components of vegetables and fruits, associated with decreasing cancer risk. A necessary first step in clarifying this conundrum is to determine whether nitrite promotes or blocks phenotypes associated with the promotion of cancer in colon epithelial cells. Data from human colon cancer cell lines representing stage 1 through 4 colon carcinomas, respectively, at were seeded into 96-well black plates and treated with vehicle control and different concentrations of nitrite for 24 or 48 hrs. Cell proliferation was measured by using standard AlamarBlue(R) assay. Our results indicate nitrite has no effect on proliferation of stage 1 SW1116 colon cancer cells at any concentration, while nitrite inhibits proliferation of stage 2 SW480 proliferation at 10nm-100microM and stage 3 HCT15 at 100nm and 1microM, but promotes a significant increase in proliferation in stage 4 COLO205 cells at 100 microM. Nitrite inhibited invasion into Matrigel(R) of stage 3 SW480 colon cancer cells in a concentration-dependent manner. However, it significantly promotes the invasion of stage 4 COLO205 colon cancer cells at the highest concentration (100 microM). Our FACS data demonstrated that nitrite decreased cell cycle progression in SW480 and HCT15 with changed G2/M transition and delayed G1 phase entry in a concentration-dependent manner. However, 100 microM nitrite promotes cell cycle progression in COLO205 cells with increased S-phase entry. Taken together, our data indicate nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas. These data may lend biological plausibility to the heterogeneous risk profiles of dietary nitrite exposure and risk of certain cancers in humans and support context-specific recommendations for consumption of dietary nitrite related to cancer risk. The manuscript describing these data are in press at Nitric Oxide. Project B: Our results demonstrate that astrocytomas and gliosarcomas OXCT1 and BDH1 at low levels while BDH2 and ACAT1 expression is maintained.
Publications
- Hord, NG. (2011)Regulation of dietary nitrate and nitrite: balancing essential physiological roles with potential health risks. In: "Nitrate and Nitrite in Human Health and Disease"; ed: NS Bryan and J Loscalzo, Humana Press.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Project A has built upon previous data and supports our hypothesis that dietary sources of nitrate and nitrite may be associated with health benefits. There is growing evidence revealing that nitrate and nitrite metabolism occurs in blood and tissue to form NO and other bioactive nitrogen oxides, representing an alternative to classical L-arginine-NO synthase -NO signaling pathway. NO is involved in neurotransmission, vasodilation, inflammation, and immunity and is also believed to play roles in multiple stages of various cancers. Our collaborative group has reported that dietary patterns high in nitrate and nitrite from vegetables and fruits may be responsible for part of the hypotensive effect of the Dietary Approaches to Stop Hypertension diet. These data have been presented at invited seminars at the University of Florida and Johns Hopkins University Bloomberg School of Public Health as well as the American Institute for Cancer Research Annual Meeting. Project B was developed using funds from the American Cancer Society, in collaboration with Drs. L. Karl Olson and Howard Chang, to determine if ketone metabolizing enzymes in brain cancer tissues are expressed, as hypothesized, at low levels. This work, titled "Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 (OXCT1) and Glial Fibrillary Acidic Protein (GFAP) Content in Human Astrocytoma and Gliosarcoma: Implications for Ketone Utilization in Cancer" was presented in August, 2010 by Ms. Courtney Meredith at an MSU undergraduate research forum. PARTICIPANTS: L. Karl Olson, Howard Chang, Courtney Meredith, Janine Ghannam (MSU) Nathan Bryan, Yaoping Tang, Pamela Berens (University of Texas) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Work on project A demonstrates that human breast milk, soy milk and dairy milks can provide a significant percentage of nitrate and nitrite intakes that approach the Acceptable Daily Intake limits of the World Health Organization (WHO). Given the cardiovascular and immunological benefits of dietary nitrates and nitrites from vegetables and fruits, these data call into question the rationale for WHO nitrate and nitrite exposure limits from vegetables and fruits. Another aim of project A was addressed in work on colon carcinoma cells in culture exposed to nitrite. Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Consumption of specific food components is associated with risk of CRC. Nitrates and nitrites, used exogenously in processed meats to enhance taste and microbial food safety and associated with increased cancer risk, are also endogenous components of vegetables and fruits, associated with decreasing cancer risk. A necessary first step in clarifying this conundrum is to determine whether nitrite promotes or blocks phenotypes associated with the promotion of cancer in colon epithelial cells. A submitted manuscript details the effect of nitrate and nitrite on cell number homeostasis in various stages of colon carcinoma cells. It exhibits both procarcinogenic and anticarcinogenic characteristics, in a concentration-dependent and carcinoma-stage-dependent manner. These in vitro data provide insights into epidemiologic observations in humans that associate nitrite intake with gastrointestinal cancer risk. We have demonstrated that nitrite differentially regulates cell cycle progression and invasive behavior in colon carcinoma cells depending upon stage. Furthermore, these findings indicate that the effect of nitrite and nitrate on cancer cells is stage-dependent and, therefore, mechanistically complex. More studies are needed address nitrite-dependent phenotypic effects in other cancer cell types and elucidate the relevant molecular mechanisms. Taken together, our data indicate nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas. These data may lend biological plausibility to the heterogeneous risk profiles of dietary nitrite exposure and risk of certain cancers in humans and support context-specific recommendations for consumption of dietary nitrite related to cancer risk. Project B results have implications for the use of ketogenic diets in the treatment of human brain cancer, specifically glioblastoma multiforme. Work continues to address the biological plausibility of treating this often fatal cancer by dietary means.
Publications
- Hord NG, Ghannam JS, Garg HK, Berens PD, Bryan NS. (2010) Nitrate and Nitrite Content of Human, Formula, Bovine, and Soy Milks: Implications for Dietary Nitrite and Nitrate Recommendations. Breastfeed Med. 2010 Oct 19. [Epub ahead of print]
- Hord, NG. (2010) Regulation of dietary nitrate and nitrite: balancing essential physiological roles with potential health risks. In: Nitrates and Nitrites in Human Health and Disease; Springer/Humana Press, Editors: Joseph Loscalzo, M.D., Ph.D (Harvard University), Nathan Bryan (University of Texas).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This project involves elucidating the physiologic determinants of nitrate and nitrite in human milk. Human breast milk confers significant nutritional and immunological benefits for the infant. Plasma nitrite has been identified as an endogenous source of nitric oxide (NO) via the action of commensal oral microbiota in adults. Nitrites have been used therapeutically in experimental models to enhance mucosal immune and cardiovascular function. The two objectives of this project are: 1) to quantify the concentration of nitrate and nitrite in human milk over the first 21 days post-partum and 2) to identify the physiologic determinants of nitrate and nitrite concentration in human milk. This report offers preliminary data on up to thirteen (13) individual donors of human milk. These preliminary results demonstrate that human milk can serve as a dietary source of nitrate and nitrite. Using sensitive quantitative methodologies, breast milk nitrate and nitrite concentrations (per hypothetical 750 ml daily intake) were estimated for: colostrum (expressed days 1-3 postpartum; n= 10), transition milk (expressed days 3-7 postpartum; n=13), and mature milk (expressed >7 days postpartum; n=11) were 1.4/ 0.60 mg, 2.83/ 0.02 mg and 2.32/ 0.07 mg, respectively. These values for nitrate concentrations were significantly higher than the nitrate concentrations in formula milk of 1.95 and 1.20 (concentrations of nitrate in two high nitrate commercial brands of formula milk; Boost Kids Essentials and Bright Beginnings Soy Pediatric). We propose to study the physiologic determinants of the nitrate and nitrite concentrations in human milk by evaluating the contributions of maternal diet, serum nitrate/nitrite concentrations, ductal epithelial nitrate and nitrite reductase activities (including mammalian- and microbiota- associated activities). PARTICIPANTS: Project collaborators are from the University the Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center, Houston, TX (N. Bryan and Y. Tang). TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We posit that breast ductal epithelia are capable of either concentrating nitrate/nitrite from serum for secretion into breast milk or that the microbiota of the ductal epithelium provide nitrate reductase activity to the host breast. The high concentration of breast milk nitrate and nitrite provide evidence for a physiologic requirement for the provision of dietary nitrite for the protection of the gastrointestinal tract in the neonate prior to the establishment of oral microbiota. The level of nitrate/nitrite supplied by breast milk is higher than that provided by commercial formula milk. The temporal relationship between the provision of nitrite in breast 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 by dietary and commensal microbiota-generated means. These data indicate that humans are adapted to consume nitrate and nitrite from birth. These results imply that dietary recommendations to limit nitrate and nitrite consumption are contrary to the composition of what has been termed "nature's perfect food".
Publications
- No publications reported this period
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