Brief Summary of Minutes of Annual Meeting

The meeting was planned, arranged and conducted by the Vice President, David Williams, Oregon State University and was held at Casa Munras in Monterey, CA. Two guests participated in the meeting as well: Dr. Siva Kolluri who is a member of a programmatically related Mega-Project interested in collaboration and Dr. Tiffany Weir who is joining W2122 as a new representative of Colorado State University.The annual meeting was called to order at 08:00 am Thursday morning, October 21, 2010 at Casa Munras in Monterey, CA. The meeting was planned, arranged and conducted by the Vice President, David Williams, Oregon State University. The meeting commenced with a brief introduction and discussion of the meeting format and agenda. That was followed by a report from the NIFA liaison, Dr. Etta Saltos and a discussion of funding and organizational changes at NIFA. After the report the administrative advisor Dr. Michael Harrington provided information on Experiment Station Section Awards for Excellence in Research. This was followed by the annual report presentations from the attending committee members and guests. The regular business portion of the meeting commenced Friday morning after the annual report presentations. Calistoga, California was selected as the site for the 2011 meeting on Thursday and Friday the 13th and 14th of October 2011. For 2011, Jim Pestka (Michigan State University) would serve as Secretary, Roger Coulombe (Utah State University) as Vice President, and David Williams (Oregon State University) as President. The responsibilities of the Rewrite Committee (Bill Helfrich, Roger Coulombe and Ron Riley) for drafting the renewal proposal was discussed. It was suggested that the committee should meet the day before the 2011 meeting (12 October 2011) to discuss the rewrite. The meeting was adjourned at around 10:00 am on 22 October.

OBJECTIVE 1. Determine mechanisms of action by which food-borne bioactive compounds protect against human diseases such as cancer, inflammation, birth defects, and microbial infection. Dr. Pratibha Nerurkar, University of Hawaii In Hawaii, Native Hawaiians and Pacific Islanders have the highest rates of obesity-associated diabetes, and Filipinos and Japanese in Hawaii have higher incidences of diabetes associated with abdominal fat accumulation. Current therapies for obesity are complicated due to factors including an inability to maintain long-term weight loss and drug-drug interactions. In addition, conventional therapies may not be affordable, suitable and/or acceptable for culturally sensitive minority populations. Momordica charantia, commonly known as bitter melon (BM) has been traditionally used in Chinese and Ayurvedic medicine, to treat diabetes and its complications. Similarly, Morinda citrifolia, commonly known as noni has been traditionally used in Hawaiian medicine, to treat diabetes and other chronic diseases. Our goal was to test the efficacy of BM and noni using cell culture and animal models. We have demonstrated earlier that BM can improve not only glucose and lipid metabolism, but also prevent weight gain in mice fed high-fat-diet (HFD) containing 58% fat. To extrapolate our studies to humans, we further tested the anti-obesity effects of BM in primary human adipocyte cell cultures. Our data indicates that BM reduced lipid accumulation and increases lipolysis in primary human adipocytes obetained commercially from overweight individuals. In a separate study we tested the anti-diabetic and anti-obesity effects of noni juice using male C57BL/6 mice fed high-fat-diet (HFD) containing 58% fat. Our data demonstrates that noni prevents weight gain and improves fasting glucose as well as glucose and insulin tolerance test in HFD-fed mice. Mechanistic studies further demonstrate that noni improves fasting glucose by inhibiting hepatic gluconeogenesis via transcription factor, forkhead box O (FoxO). Our studies help to demonstrate the efficacy of traditional medicine and identify molecular targets that can lead to drug discovery. James Pestka, Dr. Hui-ren Zhou, Brenna Flannery, Xiao Pan, Kaiyu He, Michigan State We will determine the mechanisms by which selected compounds exert their protective action. Food-borne toxins and carcinogens are present per se or are induced by processing, preparation, and other post-harvest steps. We will identify mechanisms of action and develop biomarkers of natural and induced toxicants in food for human risk assessment and disease prevention. Worldwide, obesity is a growing epidemic in need of prevention and intervention strategies. The effects of ingesting the common foodborne mycotoxin deoxynivalenol (DON) on body weight and composition were characterized in the high fat (HF) diet-induced obese mouse model. Female B6C3F1 mice were initially fed HF diets containing 45% kcal (HF45) or 60% kcal (HF60) as fat for 94 d to induce obesity. Half of each group was either continued on unamended HF diets or fed HF diets containing 10 mg/kg DON (DON-HF45 or DON-HF60) for another 54 d. Additional control mice were fed a low fat (LF) diet containing 10% kcal as fat for the entire 148 d period. DON induced rapid decreases in body weights and fat mass, which stabilized to those of the LF control within 11 d. These effects corresponded closely to a robust transient decrease in food consumption. While lean body mass did not decline in DON-fed groups, further increases were suppressed. DON exposure reduced plasma insulin, leptin, insulin-like growth factor 1 and insulin-like growth factor acid labile subunit as well as increased hypothalamic mRNA level of the orexigenic agouti-related protein. Overall, DON-mediated effects on body weight, fat mass, food intake and hormonal levels were consistent with a state of chronic energy restriction. Consumption of deoxynivalenol (DON), a trichothecene mycotoxin commonly detected in cereal-based foods, causes impaired growth in many animal species. While growth retardation is used as a basis for regulating DON levels in human food, the underlying mechanisms remain poorly understood. Consumption of deoxynivalenol (DON), a trichothecene mycotoxin commonly detected in cereal-based foods, causes impaired growth in many animal species. While growth retardation is used as a basis for regulating DON levels in human food, the underlying mechanisms remain poorly understood. Oral exposure of mice to DON rapidly induces multi-organ expression of proinflammatory cytokines and this is followed by upregulation of several suppressors of cytokine signaling (SOCS), some of which are capable of impairing growth hormone (GH) signaling. The purpose of this study was to test the hypothesis that impairment of the GH axis precedes DON-induced growth retardation in the mouse. Subchronic dietary exposure of young (4-wk old) mice to DON (20 ppm) over a period of 2 to 8 wk was found to: (1) impair weight gain, (2) result in a steady-state plasma DON concentration (40-60 ng/ml), (3) downregulate hepatic insulin-like growth factor acid labile subunit (IGFALS) mRNA expression and (4) reduce circulating insulin-like growth factor 1 (IGF1) and IGFALS levels. Acute oral exposure to DON at 0.5-12.5 mg/kg bw markedly suppressed hepatic IGFALS mRNA levels within 2 h in a dose-dependent fashion whereas 0.1 mg/kg bw was without effect. DON-induced IGFALS mRNA upregulation occurred both with and without exogenous GH treatment. These latter effects co-occurred with robust hepatic SOCS3 upregulation. Taken together, these data suggest that oral DON exposure perturbs GH axis by suppressing two clinically relevant growth-related proteins, IGFALS and IGF1. Both have potential to serve as biomarkers of effect in populations exposed to this common foodborne mycotoxin. The trichothecenes are a group of over 200 sequiterpenoid metabolites that can dysregulate cell signaling and modulate growth and immune function. Our results illustrate the dual nature of many natural chemicals in that they can promote both beneficial and adverse health effects depending on dose. Outcomes of the trichothecene mechanism studies here will include improved understanding of the molecular basis by which trichothecenes affect obesity and growth. This research will identify new targets for controlling the obesity pandemic. The data will also directly inform regulators involve in human risk assessment thus enabling better management of the risks from DON to the U.S. public. OBJECTIVE 2. Identify mechanisms of action and biomarkers of natural and induced toxicants in food for human risk assessment and disease prevention. Leonard F. Bjeldanes, Principal Investigator, Gary Firestone, Co-Investigator, UCB In our continued studies of the modes of action and potential efficacy of phytochemicals as cancer preventive and therapeutic agents, we examined the mechanism of action of Brassica indoles (I3C and derivatives) as inhibitors of cell proliferation and as modulators of estrogen receptor signaling. A. Direct Inhibition of elastase activity by indole-3-carbinol triggers a CD40-TRAF regulatory cascade that disrupts NF-ºB transcriptional activity in human breast cancer cells. Treatment of highly tumorigenic MDA-MB-231 human breast cancer cells with indole-3-carbinol (I3C) directly inhibited the extracellular elastase-dependent cleavage of membrane-associated CD40, a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 signaling has been implicated in regulating cell survival, apoptosis, and proliferation, as well as in sensitizing breast cancer cells to chemotherapy, and is therefore an important potential target of novel breast cancer treatments. The I3C-dependent accumulation of full-length unprocessed CD40 protein caused a shift in CD40 signaling through TNF receptorassociated factors (TRAF), including the TRAF1/TRAF2 positive regulators and TRAF3 negative regulator of NF-ºB transcription factor activity. Because TRAF1 is a transcriptional target gene of NF-ºB, I3C disrupted a positive feedback loop involving these critical cell survival components. siRNA ablation of elastase expression mimicked the I3C inhibition of CD40 protein processing and G1 cell cycle arrest, whereas siRNA knockdown of TRAF3 and the NF-ºB inhibitor IºB prevented the I3C-induced cell cycle arrest. In contrast, siRNA knockdown of PTEN had no effect on the I3C control of NF-ºB activity, showing the importance of CD40 signaling in regulating this transcription factor. Our study provides the first direct in vitro evidence that I3C directly inhibits the elastase-mediated proteolytic processing of CD40, which alters downstream signaling to disrupt NF-ºBinduced cell survival and proliferative responses. B. Indole-3-carbinol triggers aryl hydrocarbon receptor-dependent estrogen receptor (ER)alpha protein degradation in breast cancer cells disrupting an ER alpha-GATA3 transcriptional cross-regulatory loop. Estrogen receptor (ER)alpha is a critical target of therapeutic strategies to control the proliferation of hormone-dependent breast cancers. Preferred clinical options have significant adverse side effects that can lead to treatment resistance due to the persistence of active estrogen receptors. We have established the cellular mechanism by which I3C) ablates ER alpha expression, and we have uncovered a critical role for the GATA3 transcription factor in this indole-regulated cascade. I3C-dependent activation of the aryl hydrocarbon receptor (AhR) initiates Rbx-1 E3 ligase-mediated ubiquitination and proteasomal degradation of ERa protein. I3C inhibits endogenous binding of ER alpha with the 3-enhancer region of GATA3 and disrupts endogenous GATA3 interactions with the ER alpha promoter, leading to a loss of GATA3 and ER alpha expression. Ectopic expression of GATA3 has no effect on I3C-induced ER alpha protein degradation but does prevent I3C inhibition of ERÿý promoter activity, demonstrating the importance of GATA3 in this I3C-triggered cascade. C. 1-Benzyl-indole-3-carbinol is a novel indole-3-carbinol derivative with significantly enhanced potency of anti-proliferative and anti-estrogenic properties in human breast cancer cells. A new and significantly more potent I3C analogue, 1-benzyl-I3C, was synthesized and in comparison to I3C, this novel derivative displayed an approximate 1000-fold enhanced potency in suppressing the growth of both estrogen responsive (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cells (I3C IC50 of 52 microM, and 1-benzyl-I3C IC50 of 0.05 microM). At significantly lower concentrations, 1-benzyl-I3C induced a robust G1 cell cycle arrest and elicited the key I3C-specific effects on expression and activity of G1-acting cell cycle genes including the disruption of endogenous interactions of the Sp1 transcription factor with the CDK6 promoter. Furthermore, in estrogen responsive MCF-7 cells, with enhanced potency 1-benzyl-I3C down-regulated production of ER alpha protein, acts with tamoxifen to arrest breast cancer cell growth more effectively than either compound alone, and inhibited the in vivo growth of human breast cancer cell-derived tumor xenografts in athymic mice. Collaborations: We share samples and discuss various aspects of our work with D. Williams (OSU) and J. Pestka (MSU). Our studies have identified for the first time a molecular target for I3C provide the basis for further investigations of both the clinical usefulness of the I3C derivatives in hormone-related cancers. The studies also provide further evidence for the modes of action of I3C that will be important in the potential development of more active and less toxic pharmaceuticals based on this indole. We plan to examine the primary molecular events that are responsible for the cytostatic effects of DIM in breast tumor cells. We also plan to further characterize the immune modulation effects of DIM and mechanism of action. We will screen fungi and plants from Indonesia for activities against cancer, immune disorders and neurological problems. The results of our cell culture studies continue to support the notion that certain indoles present in food plants will be useful in the control of some cancers and other hormone related disorders. RONALD T. RILEY, K. Voss, N. Zitomer, T. Glenn, C. Bacon, USDA, Athens, GA Exposure and mechanism-based biomarkers are needed to determine if fumonisins are contributing factors in human disease. ARS researchers in Athens, Georgia have developed methods that will be used in Guatemala to measure urinary fumonisin in humans and lipid biomarkers in blood spots. A human subject's research protocol was also approved for a study in the USA to validate our methods for fumonisin in urine and sphingoid base 1-phosphates in blood spots. These studies have revealed that only fumonisin B1 is excreted in urine even though other fumonisins are approximately 30% of the total fumonisin consumed. We found that the amount of fumonisin B1 excreted is less than 1% of the dose and that it is cleared from the urine very rapidly (<96 h). A method was also developed for measuring sphingoid base 1-phosphates in blood spots. Studies were conducted to determine the limits of detection, recoveries and stability of samples in blood spots collected on FTA elute cards and stored dessicated at -20oC. The findings from the extrusion study described under objective 4 below have also provided additional information on the relationships between sphingolipid metabolism disruption and toxicity. Both microscopic pathology scores and total Sa concentrations were tightly correlated with the daily FB1 intakes. It is also noteworthy that deoxy Sa was not found in the livers or kidneys. Deoxy Sa, which is produced when alanine is substituted for serine in the first step of de novo sphingolipid biosynthesis, accumulated in the livers of mice following exposure to FB1 via the diet or intraperitoneal injection. The absence of deoxy Sa in rat liver suggest that sphingolipid metabolism in rats and mice differ in some respects and that these differences might influence their target organ-specific responses to fumonisin exposure. Additional studies are warranted to more thoroughly characterize sphingolipid metabolic responses to FB1 in these and other species and to better understand the relationships between disrupted sphingolipid metabolism and nephrotoxicity. Fumonisin B1 (FB1) is found in maize and maize-based food and is a suspected risk factor for cancer or birth defects in humans. Fusarium verticillioides fermented maize grits (Batch-1= 9.7 ppm FB; Batch-= 50 ppm) were extruded without (Batch-1E; Batch-2E) or with 10% glucose supplementation (Batch-1EG; Batch-2EG). FB1 concentrations after cooking were: Batch-1E = 2.7 ppm; Batch-1EG = 0.6 ppm; Batch-2E = 18 ppm; and Batch-2EG = 5.7 ppm: FB1 concentrations were reduced 64% (Batch-2E) to 94% (Batch-1EG). The raw and processed grits were fed (50% w/w in rodent chow) to rats for up to 8 weeks. FB1 intakes averaged 354, 103, and 25.1 µg/kg body weight/day for Batch-1, Batch-1E and Batch-1EG and 1804, 698, and 222 µg/kg body weight/day for the Batch-2, Batch-2E and Batch-2EG, respectively. Dose-dependent kidney effects included apoptotic lesions and elevated sphingoid base concentrations found in the groups fed Batch-1, Batch-1E, Batch-2, Batch-2E, or Batch-2EG. In contrast, kidney lesions and sphingoid base elevations were absent in the Batch-1EG group. Therefore, extrusion with glucose supplementation reduced FB1 concentrations in contaminated maize grits to levels that were not toxic in vivo. Developing exposure and mechanistic markers provides us with a tool to pinpoint high risk populations for the studies in humans and will aid us in our goal to reveal the factors (genetic and nutritional) leading to possible increased susceptibility to neural tube defects in populations where maize consumption is high and diets are likely to be deficient in folate. These studies are critical if we are to develop an understanding of fumonisin potential for involvement in human disease. Demonstrating that extrusion cooking alone or with sugar supplementation reduces fumonisin concentrations and in vivo toxicity improves confidence in the efficacy of extrusion for reducing fumonisins without the production of unknown, toxic fumonisin breakdown products. OBJECTIVE 3. Discover bioactive compounds that have beneficial or adverse effects on human health. Bill Helferich, UNIVERSITY OF ILLINOIS, URBANA (Dr. Judy Yang, Jim Hartman, Wendan Wang) Soy isoflavones, genistein and daidzein, are widely consumed in soy-based foods and dietary supplements for their putative health benefits; however, evidence for potential adverse effects has been obtained from experimental animal studies. An important prerequisite for understanding the pharmacodynamics of isoflavones is better information about pharmacokinetics and bioavailability. This study determined the bioavailability of genistein and daidzein in a mouse model by comparing plasma pharmacokinetics of their aglycone and conjugated forms following administration of identical doses (1.2 mg/kg genistein and 0.55 mg/kg daidzein) by either an intravenous injection (IV) or gavage of the aglycones in 90% aqueous solution vs a bolus administration of equimolar doses delivered in a food pellet prepared using commercial soy protein isolate (SPI) as the isoflavone source. The bioavailability of genistein and daidzein was equivalent for the gavage and dietary routes of administration despite the use of isoflavone aglycones in the former and SPI-derived glucosides in the latter. While absorption of total isoflavones was nearly quantitative from both oral routes [>84% of areas under the curve (AUCs) for IV], presystemic and systemic phase II conjugation greatly attenuated internal exposures to the receptor-active aglycone isoflavones (9-14% for genistein and 29-34% for daidzein based on AUCs for IV). These results show that SPI is an efficient isoflavone delivery vehicle capable of providing significant proportions of the total dose into the circulation in the active aglycone form for distribution to receptor-bearing tissues and subsequent pharmacological effects that determine possible health benefits and/or risks. Usefulness of Findings: Tools to study metastasis are limited and here we have used bioluminescence imaging to follow metastasis of a highly metastatic tumor cell line (4T1) from a mouse mammary cancer. The findings presented here indicate that BLI can be used to effectively follow metastatic progression in an animal model. MICHAEL S. DENISON, UNIVERSITY OF CALIFORNIA, DAVIS (Dr. B. Zhao, Dr. G. He, Dr. A. Hayashi) The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that plays a key regulatory role in endogenous developmental and physiological processes and also is responsible for mediating the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs). In addition, agonists/antagonists of the AhR have been shown to exert a variety of anticarcinogenic effects and as such, it has become a target for development of therapeutic agents. Studies from many laboratories have demonstrated that the AhR can bind and be activated by a structurally diverse range of synthetic and naturally-occurring chemicals (including endogenous chemicals), but the lack of crystal structure of the AhR ligand binding domain (LBD) has prevented mechanistic analysis to attempt to explain this binding diversity. Site-directed mutagenesis and ligand binding/activation studies and more recent improvements in our AhR LBD structural homology model, have provided fairly convincing evidence that while structurally distinct AhR agonists produce a similar activation effect on the AhR, they appear to differentially interact with amino acids within the AhR ligand binding pocket. This differential binding hypothesis has been supported by: 1) mechanism-based common reactivity pattern (COREPA) modeling of structurally diverse AhR ligands, 2) demonstration of the ability of mutations of selected amino acids within the AhR LBD to differentially affect the binding and activation of the AhR by various ligands, and 3) our identification of a novel AhR antagonist (CH23191) that can specifically antagonize some AhR ligands (i.e. TCDD and related halogenated aromatic hydrocarbons), but not other ligands (i.e. beta-naphthoflavone, a variety of polycyclic aromatic hydrocarbons (PAHs) and other PAH-like AhR agonists). Taken together, we these results provide key insights into the mechanisms contributing to the demonstrated structural diversity of AhR ligands and raise questions as to whether these differential interactions could contribute to ligand-dependent differences in AhR-dependent toxic and biological effects. We have also improved and expanded our recombinant cell-based bioassay systems for detection of AhR agonists and these improved third generation (G3) cell bioassays have a 10-50-fold increased sensitivity and produce more than a 100- to 1,000-fold greater induction response to AhR agonists. These improved G3 cell bioassays have the sensitivity needed for screening of food samples for low level contamination of dioxin and dioxin-like chemicals. These improved bioassays also have significant application for high-throughput screening to identify novel AhR agonists and antagonists that have potential therapeutic applications. We have developed and optimized an improved recombinant human cell G3 bioassay for detection and characterization of human AhR ligands (agonists and antagonists). This new bioassay has been used in a variety of applications. It has been used to identify a novel human-specific AhR antagonist that can promote the expansion of human hematopoietic stem cells and thus has significant therapeutic potential. In addition, our human G3 cell bioassay has been used for high-throughput screening of >300,000 compounds from an NIH chemical library to identify AhR agonists (as part of a NIH X01 grant) and initial screening has identified >100 novel compounds that appear to be specific for the AhR (they failed to activate another promiscuous ligand-activated receptor (i.e. the pregnane X receptor (PXR)). These compounds are being further characterized for their ability to specifically activate the AhR and AhR-responses. Our analyses of the promiscuity of AhR ligand binding has revealed significant differences in the ability of HAHs (i.e. dioxin and dioxin like compounds) to bind to the AhR in a manner distinctly different than that of nontoxic AhR agonists (i.e. flavonoids, indirubins and PAHs). These observations could suggest that differences in how these toxic and non-toxic AhR ligands bind to the AhR could contribute to their differential ability to produce beneficial and/or adverse effects in exposed animals. We have identified and characterized the ability of CH223191 to selectively antagonize the ability of toxic HAHs to bind to and activate the AhR, with little or no effect on PAHs and other nontoxic AhR ligands. CH223191 is the first ligand-selective AhR identified to date and given its selectivity, it has potential as a therapeutic agent (or lead compound to generate a therapeutic agent) that can block the toxicological and biological effects of toxic HAHs, while not inhibiting the ability of non-toxic/beneficial AhR ligands to bind to and activate the AhR. Our improved third generation recombinant cell-based bioassay systems for detection of AhR agonists are currently being evaluated and subjected to validation studies by the European Union for adoption as an EU approved screening method for dioxins and related HAHs in food. We will continue our characterization and analysis of the binding of naturally-occurring and synthetic chemicals to the AhR by high-throughput gene expression analysis using our improved cell gene expression bioassay systems, molecular docking studies using our improved AhR LBD homology model, and in vitro ligand and DNA binding analysis. Structure-activity relationship analysis of novel AhR agonists (from our high-throughput screening analysis) as well as antagonists (CH223191, its derivatives and several other promising compounds) will also be carried out using AhR-based in vitro and cell-based bioassays as well as molecular docking studies to further understand the mechanisms by which structurally diverse chemicals bind to the AhR LBD to produce agonist or antagonist responses. In addition, we will examine the mechanisms responsible for species-specific antagonist activity of several newly identified compounds. Donato F. Romagnolo, Andreas J. Papoutsis, Ornella Selmin, Jamie Borg, University of Arizona The BRCA-1 protein is a tumor suppressor involved in repair of DNA damage. Epigenetic mechanisms contribute to its reduced expression in sporadic breast tumors. Through diet, humans are exposed to a complex mixture of xenobiotics and natural ligands of the aromatic hydrocarbon receptor (AhR), which contribute to the etiology of various types of cancers. The AhR binds xenobiotics, endogenous ligands, and many natural dietary bioactive compounds, including the phytoalexin resveratrol (Res). In estrogen receptor- alpha (ER alpha )-positive and BRCA-1 wild-type MCF-7 breast cancer cells, we investigated the influence of AhR activation with the agonist 2,3,7,8 tetrachlorobenzo(p)dioxin (TCDD) on epigenetic regulation of the BRCA-1 gene and the preventative effects of Res. We found that activation and recruitment of the AhR to the BRCA-1 promoter hampers 17 beta -estradiol (E2)-dependent stimulation of BRCA-1 transcription and protein levels. These inhibitory effects are paralleled by reduced occupancy of ER alpha, acetylated histone (AcH)-4, and AcH3K9. Conversely, the treatment with TCDD increases the association of mono-methylated-H3K9, DNA-methyltransferase-1 (DNMT1), and methyl-binding domain protein-2 with the BRCA-1 promoter and stimulates the accumulation of DNA strand breaks. The AhR-dependent repression of BRCA-1 expression is reversed by small interference for the AhR and DNMT1 or pretreatment with Res, which reduces TCDD-induced DNA strand breaks. These results support the hypothesis that epigenetic silencing of the BRCA-1 gene by the AhR is preventable with Res and provide the molecular basis for the development of dietary strategies based on natural AhR antagonists. This was an exploratory study developed to investigate the opportunities for targeting of cyclooxygenase-2 (COX-2) expression with bioactive food components for the prevention of colon cancer. Factors related to diet and life style have been identified as primary determinants in about 80% of colorectal cancers. Non-steroidal anti-inflammatory drugs (NSAID) and selective (COX-2) inhibitors (COXIB) reduce the relative risk of colon cancer. To overcome systemic COX inhibition associated with NSAID and COXIB, there is a growing interest in developing alternative colon cancer prevention strategies using diet-based approaches that target COX-2. The transition from aberrant crypt foci (ACF) to colon cancer is a multiyear process providing opportunities for nutritional targeting of genes influencing the course of this disease process at early stages of development. The activation of the proinflammatory gene COX-2 and PG production in the colonic mucosa are recognized risk factors in colon cancer. Many natural food components may impact colon cancer risk by interfering with ligand-activated receptors, signal transduction pathways, and transcription factors involved in stimulation of COX-2 expression. In a systematic review of published studies, we highlighted key upstream features of signaling pathways and transcriptional control of the COX-2 gene and discussed opportunities for dietary modulation of COX-2 expression in gastro-intestinal cancers with special emphasis on prevention of colorectal tumors. Review of the experimental evidence suggests that dietary strategies based on specific or cocktails of bioactive food components as well nutritional-pharmacological combinations targeted to regulation of COX-2 expression and activity may prove useful in the prevention of colon cancer. An integrated approach may offer the advantage of combined higher efficacies. Future studies should investigate the efficacy of combinations of bioactive food compounds on epigenetic regulation of the COX-2 gene and characterize potential synergies and amplification effects resulting from the concomitant use of bioactive food components and COX-2 inhibitors. We continue our work related to epigenetic silencing of tumor suppressor genes induced by the AhR and are currently developing animal models to test this hypothesis in vivo. OBJECTIVE 4. Develop strategies to increase beneficial or decrease adverse effects of bioactive food constituents and microbial contaminants. David E. Williams, Roderick Dashwood, Robert Tanguay, Oregon State University This program will continue to focus on mechanisms of cancer prevention by food now with addition funding from the National Cancer Institute to test phytochemicals from cruciferous vegetables (indole-3-carbinol and sulforaphane) and compare the efficacy of these dietary supplements to the whole foods from which they were derived. The focus will be on colon, prostate and transplacental cancer (lymphoma, lung and liver). Studies are also underway to further investigate mechanisms of action of mycotoxins in food, primarily aflatoxin B1, the potential for this mycotoxin to cross the placenta and cause liver cancer later in life in the offspring and how we can supplement the mothers diet to provide protection from that exposure. The program is now partnering closer with The Linus Pauling Institute at Oregon State University to maximize the impact of the research and we have a new emphasis on the exciting area of epigenetics. In summary, this program seeks to identify, understand, and eliminate, in so far as practicable, the specific toxicants in the food supply that contribute to health deficits as well as mechanisms of agent for dietary chemopreventive, beneficial food components. The addition of high-throughput assays utilizing zebrafish embryos allows us to screen many different natural products for beneficial effects against development toxicants. Dr. Dashwood has continued to study how the isothiocyanate phytochemical from cruciferous vegetables, sulforaphane, acts as an HDAC (histone deacetylase) inhibitor. HDACs are very important in gene regulation as deacetylation of histone tends to compact the structure of DNA and inhibit transcription. There are numerous HDAC inhibitors, some of which are now being used to treat human disease including cancer. HDAC inhibitors tend to maintain a relatively open chromatin structure enhancing transcription. Dr. Dashwood has determined that the metabolites of sulforaphane are much more potent HDAC inhibitors than the parent compound. He is applying this knowledge to enhance our understanding of the most efficient ways to inhibit cancer (primarily colon) through diet. Dr. Williams is continuing with his work on the transplacental model of cancer chemoprevention. Work underway will determine whether pure phytochemical or whole food is more effective in protection of the fetus from carcinogens that can cross the placenta, when is the critical time of exposure for the fetus for maximum protection. In collaboration with Dr. Dashwood, the mechanism of this protection is under study, i.e., is the mechanism of protection epigenetic? If so, is it possible to carry this protection through to the F3 (great-grandchildren) generation? Both Dr. Williams and Dr. Dashwood are initiating chemoprevention studies with cruciferous vegetables with human volunteers. This enhances the translational nature of the research and the impact on human health. There have been many outcomes to our research in chemoprevention by diet. We have published this work in quality journals, been invited to speak at national and international meetings as well as academic institutions. The popular press has also found this story to be very interesting. In 2010, Drs. Dashwood and Williams (along with Dr. Emily Ho from Nutrition and Food Management) obtained 6.8 million dollar, five year grant from the National Cancer Institute of the NIH to continue these studies.

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