Brief Summary of Minutes of Annual Meeting

W-1122: Beneficial and Adverse Effects of Natural, Bioactive Dietary Chemicals on Human Health and Food Safety Annual meeting of the technical committee, October 12-14, 2011 Calistoga, CA The annual meeting was called to order at 0830 Thursday morning, October 13th, 2011. Advisor Michael Harrington provided a report on the updated priorities and administrative structure of USDA NIFA as well as changes to AFRI. He also presented budget projections for FY 2012. Because of the upcoming project renewal the remainder of Dr. Harringtons remarks was focused on determining and communicating the impact of our efforts. This included defining outputs versus impacts and discussion on writing impact statements. After Dr. Harrington's comments the annual reports were given as follows: Technical Reports Williams, Dave - Oregon State University Bjaldanes, Len - University of California at Berkeley Coulomb, Roger - Utah State University Weir, Tiffany - Colorado State University Helferich, William - University of Illinois Nerurkar, Prathiba - University of Hawaii Pestka, James - Michigan State University Friedman, Mendel - USDA-WRRL Denison, Michael - UC Davis Ricketts, Marie-Louise  University of Nevada Writing assignments and revision of the new project plan for the next five years were discussed and changes were made by the group to the draft document. Newly written sections will be submitted to Dr. Coulomb who will compile the final proposal for submission. The W2122 project plan is due January 2012. The regular business portion of the meeting commenced at 0830 on Friday, October 14th. Estes Park, Colorado was selected as the site for the 2012 meeting. For 2012, Marie-Louise Ricketts was selected to serve as Secretary, Tiffany Weir as Chair-elect and James Pestka as Chair. The meeting was adjourned at around 1200.

Accomplishments ACCOMPLISHMENTS FOR THE ENTIRE SPAN OF THE PROJECT ARE SUMMARIZED BEGINNING WITH 2011 The activities described below have addressed specific components of the W2122 Multistate project as outlined in the milestones document for the project. Many of the activities have been collaborative efforts among W2122 participants and all have benefited from the critical review provided by all participants at the annual meetings and throughout the year. Over the five year span of the project, the activities described below have been documented by approximately 300 publications and numerous reports and data provided to industry, consumers, and authoritative bodies. Participants have also disseminated results at meetings of professional societies such as the Phytochemical Society of North America, Society of Toxicology, and at Keystone meetings and Gordon conferences. OBJECTIVE 1. Consumption of food-borne bioactive compounds can protect against human diseases such as cancer, inflammation, birth defects, and microbial infection. We will determine the mechanisms by which selected compounds exert their protective action. 2011 In collaborative efforts with multiple entities, Dr. Friedman at USDA, ARS, Albany found that mushroom extracts inactivated leukemia cells in vitro and reduced tumor size in tumor bearing mice. They also found that rice hull smoke extracts inactivated Salmonella in apple juice and protected infected mice against mortality and alloxan-induced diabetic mice against diabetes. Similarly, carvacrol, the main ingredient of oregano oil, and cinnamaldehyde, the main ingredient of cinnamon oil, facilitated thermal destruction of multiple Salmonella strains in ground chicken. The Albany group and collaborators also found that the olive compound hydroxytyrosol inactivated both Staphylococcus aureus bacteria and the Staphylococcus enterotoxin A (SEA) and that an olive extract inhibited Salmonella bacteria on organic leafy greens. An olive extract and onion powder also inactivated E. coli O157:H7 and inhibited the formation of carcinogenic heterocyclic amines in grilled ground beef patties. The group in Hawaii demonstrated that noni juice improves glucose and lipid metabolism, and prevents weight gain in high-fat-diet (HFD fed mice. Their work indicates a role for chronic inflammation in the pathophysiology of obesity and Type 2 diabetes, and suggests that noni improves HFD-associated systemic inflammation by reducing secretions of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFa), interferon-gamma (INFg), interleukin 6 (IL-6), and hepatic inflammation. Noni is a Polynesian ethnomedicine, and may be used to alleviate health disparities among culturally sensitive populations such as Native Hawaiians. At Colorado State University, researchers are conducting a clinical dietary intervention (called BENEFIT) in a colon cancer survivor population to assess the potential for bioactive rice bran and bean phytochemicals to increase the levels of beneficial microbes present in the gut microbial community resulting in modulation of mucosal immune responses and the increased the production of anti-tumerogenic and anti-inflammatory microbial metabolites such as butyrate and other short chain fatty acids (SCFAs). They have identified a target community of potentially harmful and beneficial microbes and are assessing how they may be altered by dietary rice bran or dry bean. These results are important because they could result in new dietary recommendations for colon cancer prevention and identify microbial and metabolite biomarkers that could be used to assess risk or evaluate responses to a particular intervention. 2007 to 2010 Researchers from UC-Berkeley examined the mechanism of action of Brassica indoles (indole-3-carbinol and derivatives) as inhibitors of cell proliferation and as modulators of estrogen receptor signaling and determined that 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. Indole-3-carbinol also triggers aryl hydrocarbon receptor-dependent estrogen receptor (ER)a protein degradation in breast cancer cells disrupting an ERa-GATA3 transcriptional cross-regulatory loop. They also identified 1-Benzyl-indole-3-carbinol as a novel indole-3-carbinol derivative with significantly enhanced potency of anti-proliferative and anti-estrogenic properties in human breast cancer cells. The group from the University of Hawaii examined bitter melon (BM) and noni , two plants used in traditional Polynesian medicine, for anti-obesity and anti-diabetic effects in mouse models. They determined that both plants improved glucose and lipid metabolism and prevented weight gain. BM reduced lipid accumulation and increased lipolysis in primary human adipocytes obtained commercially from overweight individuals and lowered plasma apolipoprotein B (apoB) by improving hepatic insulin receptor phosphorylation and its downstream signaling molecules. Noni improved fasting glucose by inhibiting hepatic gluconeogenesis via transcription factor, forkhead box O (FoxO). These studies help to demonstrate the efficacy of traditional medicine and identify molecular targets that can lead to drug discovery. 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. A W2122 team from the University of Arizona found that epigenetic silencing of the BRCA-1 breast cancer gene by the AhR is preventable with resveratol and provides a molecular basis for the development of dietary strategies based on natural AhR antagonists. They determined that AhR directly participates in the trans-activation of the COX-2 promoter and that this mechanism is a potential target for dietary agents in the prophylactic management against stimulation of COX-2 expression by AhR agonists. The Utah State W2122 researchers developed poultry as a model for sensitivity to dietary carcinogens, like aflatoxin B1 (AFB1), and responsiveness to chemopreventives such as phenolic antioxidants. This model offers several advantages over traditional rodent models because the turkeys lack detoxifying glutuathione S-transferases (GSTs) and efficiently bioactivate AFB1 to the reactive exo-AFB1-8,9-epoxide (AFBO) . The team at Oregon State identified that chlorophyllin and indole-3-carbinol were very effective in inhibition of transplacental carcinogenesis due to administration of the polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP) to pregnant mice. The group at University of Illinois found that breast cancer patients with tamoxifen treatment consume considerable isoflavone supplements, and that the potential combined effect of tamoxifen and genistein, present in isoflavone supplements, may result in significant undesirable outcome on mammary tumors. W2122 researchers at Michigan State found that trichothecene mycotoxin deoxynivalenol (DON) induced interleukin-6 (IL-6)-dependent IgA nephropathy (IgAN) in mice can be prevented by feeding long chain n-3 polyunsaturated fatty acids (PUFAs) found in fish oil. DON-induced IL-6 expression is CREB-mediated and PKR-dependent and the requisite kinase activities for these pathways were suppressed in macrophages from mice fed docosahexaenoic acid (DHA) for an extended period. Scientists from USDA-ARS, Albany collaboratively developed a computational model that revealed that the number of hydrogen bonds formed by seven tea catechins with simulated cell membranes correlates with our published observations on relative antimicrobial and inhibitory effects of the same catechins against Bacillus cereus and human cancer cells, respectively. Computational modeling of catechin-cell membrane relationships contribute to understanding of molecular mechanisms of antimicrobial and anticancer activities of catechins. OBJECTIVE 2. 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. 2011 The Utah group has utilized their poultry (turkey) model for testing extreme sensitivity to dietary carcinogens, like aflatoxin B1 (AFB1), and responsiveness to chemopreventives. They used a genomic approach and biochemical characterization of GSTs aimed at identifying genetic markers related to AFB1 susceptibility and resistance in turkeys. They also identified P450 1A5 as the dominant enzyme responsible for AFB1 bioactivation and metabolism at environmentally-relevant AFB1 concentrations in turkey liver. These results provide a basis for breeding for increased resistance to aflatoxins in farm raised turkeys. The group at Michigan State assessed the effects of ingesting the foodborne mycotoxin, deoxynivalenol (DON), on body weight and composition in a high fat (HF) diet-induced obese mouse model. They found that DON induced rapid decreases in body weights and fat mass of but not in decline of lean mass. DON exposure also 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. The group at Michigan State also devised a short-term mouse model to investigate induction of food refusal by DON. They found that transient and DON dose-dependent induction of anorexia occurred within 2 h of exposure. This short-term mouse bioassay was useful in characterizing DON-induced anorexia and should be applicable to elucidating mechanisms underlying this adverse nutritional effect. The USDA-ARS, Athens group has collaborated with scientists at the University of Nebraska Medical Center to determine the possible role of fumonisin(FB)-induced elevation of sphingoid base 1-phosphates, ligands for S1P Receptors (S1P1-5) as a risk factor for neural tube defects in mice. They are also exploring urinary FB as an exposure marker and sphingolipid base 1-phospatase as a mechanistic marker. The results strongly suggest that the lysophospholipid receptors known as S1PR could play a key role in fumonisin-induced NTD in mice. This is an important discovery and provides a potential human risk factor which is being explored in a human study conducted in Guatemala. 2007 to 2010 W2122 researchers at Michigan State found that oral DON exposure perturbs the growth hormone 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. They also found that young mice are modestly more susceptible than adult mice to the adverse effects of DON and that this might result from a greater toxin tissue burden. The Michigan State group has also identified mechanisms related to the molecular affects of trichothecene on obesity and growth. This research will identify new targets for controlling the obesity pandemic and will directly inform regulators involved in human risk assessment thus enabling better management of the risks from DON to the U.S. public. This group has also studied translational inhibitors such as DON and ribosomal inactivating proteins (RIPs), which induce mitogen-activated protein kinase (MAPK)-driven chemokine and cytokine production by a mechanism known as the ribotoxic stress response (RSR). They found that ricin cleaves 28S rRNA directly and indirectly via RNase induction whereas DON induces 28S rRNA cleavage only by indirect induction of RNases. They also found that double-stranded RNA-activated protein kinase plays a common role in IL-8 induction by DON and two RIPs, suggesting that this kinase might be a critical factor in ribotoxic stress response. The group at USDA-ARS, Athens identified lysophospholipid receptors known as S1PR that could play a key role in fumonisin-induced neural tube defects in mice. This is an important discovery and provides a potential human risk factor which could be explored in translational studies. They also developed a questionnaire to identify heavy consumers of maize products in that can be utilized in human trials and a method to measure urinary fumonisin and lipid biomarkers in blood spots. They also made progress in characterizing sphingolipid metabolic responses to FB1 to better understand the relationships between disrupted sphingolipid metabolism and nephrotoxicity. Researchers from Oregon State have identified Cyp1b1 as a new gene target for chemoprevention of PAH-induced transplacental cancers. These studies have identified a critical window for prevention efficacy. This group also provided the most complete pharmacokinetic analysis ever conducted for AFB1 in humans, and the first evidence that natural chlorophyll can reduce systemic uptake of AFB1 from the GI tract in humans. The Illinois group showed that the dietary supplement Avlimil, containing 11 different botanicals, had either estrogenic or anti-estrogenic properties depending on the dietary dosage, suggesting that Avlimil may not be safe for women with estrogen-dependent breast cancer. OBJECTIVE 3. Selected classes of bioactive compounds show potential for beneficial or adverse effects on human health. We will discover bioactive compounds that have beneficial or adverse effects on human health. 2011 As part of a large multi-national study, the group at UC-Berkeley has been evaluating microbial and plant sources from Indonesia for anticancer, immune modulating and neurological activities and to provide in vitro bioassay support for discovery of the active components. They continue to employ cell-based assays for cytotoxicity in cultured tumor cells, NF-kB activation or inhibition in cultured macrophages, and opioid G-protein coupled receptor (GPCR) activation or inhibition in cultured human kidney cells as well as conducting follow-up studies in rodents. They have identified a number of bioactive microbial extracts that show selective cytotoxicity in breast cancer cell line MDA-MB-231 and against PC3 and LNCaP prostate tumor cells. An isolated mixture of two flavonoids-like compounds was identified to have cytostatic activity against MCF-7 cells. The UC-Berkeley group has also identified a number of lead fractions that show potent anti-inflammatory activity. Fraction H29 was highly active in the RAW-KB assay with low cytostatic activity. One compound was identified as penicillic acid (PCA) and strongly induced phosphorylation of Erk1/2, possibly activating the formyl peptide receptor that activates expression of TNF-a. Other leads are being tested for dose-response and being subjected to further chemical analysis for compound identification. The team at UC-Davis has continued to utilize recombinant human cell lines containing an aryl hydrocarbon receptor (AhR)-responsive luciferase gene as a high throughput screen to identify anti-carcinogenic AhR agonists. They have screened a chemical library of >324,000 compounds (counter screened with a pregnane X-receptor cell bioassay) and identified 108 AhR-selective agonists. These compounds have been subsequently screened for their ability to directly bind to and activate the AhR and AhR-dependent gene expression and also to act as both activators and inhibitors of estrogen receptor-dependent gene expression and cell proliferation. Several candidate compounds have been identified and are currently being modified for structure activity relationship analysis to optimize the structure for antiestrogenic functional characteristics. UC-DAVIS group has also refined a homology model of the ligand binding domain (LBD) for both analysis of the binding of ligands and ligand-dependent AhR activation. This has resulted in improvements to the model and they are now beginning docking analysis of ligands with the AhR LBD and to use this information to predict binding characteristics of structurally diverse ligands. Results generated by modeling have provided insights into how structurally diverse ligands can bind to the AhR LBD, but still activate the AhR by a common mechanism. The UC-Davis group has continued to enhance and improve their cell bioassay systems for AhR. These developments include amplification of the number of AhR DNA binding sites in the luciferase reporter plasmid and stable transfection of this vector into cell lines and the generation of mouse, rat, human and guinea pig third generation recombinant cell lines. These cell lines have been optimized to identify contamination by dioxin-like chemicals in food screening. The Colorado State University group is also examining the bioactive components from a fermented Chinese tea that has shown cytotoxic effects against colon cancer cell lines HT-29 and CaCo-2 as well as antimicrobial activity against Staphylococcus aureus and Shigella sonnei. They have identified novel fatty acid amines that likely originate from the fermenting fungus and may contribute to this teas bioactivity in vitro. The team from Illinois explored the effects of letrozole on breast cancer metastasis in a rodent model. They developed a BC metastasis model in which mammary tumor cells are injected in to the marrow cavity and metastasis from bone to lung is followed by bioluminescent imaging (BLI). Their goal was to study the effects of letrozole on BC metastasis from bone to lung in mice inoculated with murine 4T1 cancer cells. They observed that there were significantly fewer tumors on the lungs in mice in the Letrozole group compared with the Intact Control group. Letrozole was also effective to inhibit cancer cell proliferation in lungs indicated by Ki-67 staining. As a conclusion, letrozole may inhibit BC metastasis in mice inoculated with murine 4T1 cancer cells, especially in intact mice. 2007 to 2010 The research team at UC-Davis 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. This group also identified twenty synthetic flavonoids relatively potent AhR agonists, several with relative potencies in the nM range. The ability of these chemicals to inhibit the proliferation of human hepatocarcinoma and breast cancer cell lines, but not those of other AhR-containing cancer cell lines (A431) demonstrated their potential as chemotherapeutic activity in selected cancer cell types. The identification of a novel chemical that can act as an antagonist of the ability of the most potent ligand of the AhR, namely that of the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but not that of the flavonoids, provided strong support for differential ligand binding of chemicals to the AhR. The UC-Davis team has developed a three-dimensional homology model of the mouse AhR (mAhR) PAS B ligand binding domain by extending this analysis using comparative structural modeling studies of the ligand binding domains of six additional high affinity mammalian AhRs. These results, coupled with site directed mutagenesis and AhR functional analysis, has allowed detection of the "TCDD-binding fingerprint" of conserved residues within the ligand binding cavity necessary for high affinity TCDD binding and TCDD-dependent DNA binding. Researchers from Oregon State have determined that the metabolites of sulforaphane are much more potent histone deacetylase inhibitors than the parent compound. They are applying this knowledge to enhance understanding of the most efficient ways to inhibit cancer (primarily colon) through diet. The UC-Berkeley team found that an extract of 22 herbs, MF101, is an estrogen receptor beta agonist. They also isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERb but not ERa. These results demonstrate that some plants contain highly selective estrogens for ERb. They also demonstrated that DIM binds to the oligomycin binding site on the F1 transmembrane component on mitochondrial F1F0-ATPase and is an ERb-selective compound that might be an alternative to estrogens that are used in hormone therapy, which non-selectively activate both ER subtypes and are associated with increased breast cancer risk. They also showed that DIM can decrease the accumulation and activity of the key angiogenesis regulatory factor, HIF-1a, in hypoxic tumor cells. N-Alkoxy derivatization of indole-3-carbinol (I3C) increases the efficacy of the G1 cell cycle arrest of human MCF-7 breast cancer cells. The group at UC-Berkeley established the first I3C structure activity relationship for cytostatic activities, and implicates I3C-based N-alkoxy derivatives as a novel class of potentially more potent experimental therapeutics for breast cancer. The W2122 research team from Hawaii found that momordicosides and triterpenoids from bitter melon specifically demonstrated hypoglycemic effects in diabetic mice, but there appears to be a synergistic effect with the whole food having more activity than the fractionated components. OBJECTIVE 4. Modifying foods is an increasingly important strategy to improve nutrition and safety. Therefore, we will improve food safety by developing approaches to increase beneficial or decrease adverse effects of bioactive food constituents and microbial contaminants. 2011 The group at Colorado State University has explored how the bioactivity of certain foods may be potentiated through microbial fermentation. Often anti-oxidant flavonoids and other bioactive chemicals are attached to plant cell wall matrices or are present as poorly absorbed glycosides. Fermentation of foods pre-consumption may enhance both the amount and bioavailability of these compounds by converting them to more easily absorbed aglycone forms or releasing them from ligno-cellulose matrices. The CSU team determined that Sacchromyces boulardii-fermentation of rice bran alters its phytochemical profile and increased cytotoxicity against Raji lymphoma cells and Caco-2 and HT-29 colon cancer cell lines. In vivo pharmacokinetics to study bioavailability of rice bran components are underway. The group from USDA-ARS, Albany has developed edible fruit and vegetable based anti-microbial films that can be used to mitigate the presence of food-borne pathogens like E. coli and Salmonella on meat products. The team from USDA-ARS, Athens has identified mechanisms of fumosin translocation and disruption of sphingolipid metabolism in maize seedlings. Plant debris in fields can be a significant source of fumonisins and thus the factors which control fumonisin entry into plant parts could reduce the levels of fumonisins in field debris. These results are important because they could result in new strategies for resistance to Fusarium verticilliodes maize seedling diseases. 2007 to 2010 The USDA-ARS Athens group demonstrated that extrusion cooking alone or with sugar supplementation reduces fumonisin concentrations and in vivo toxicity, improving confidence in the efficacy of extrusion for reducing fumonisins without the production of unknown, toxic fumonisin breakdown products. The USDA-ARS, Albany group have found that apple based edible antimicrobial films wrapped on contaminated ham and stored at 23 or 4°C for 72 hrs induced multi-log reductions in Listeria monocytogenes. In a collaborative study we also found that apple and tomato based edible films containing the plant essential oils from allspice, bay leaf, clove bud, thyme, and vanilla inactivated E. coli O157:H7 both in direct contact and in the vapor phase. These finding support the possible commercial use of the films and indicate that they may provide antimicrobial benefits, even when not in contact with the food. They also discovered that the addition of a polyphenol-rich apple skin powder to contaminated ground beef reduced by up to two-thirds the time needed to heat-inactivate E. coli O157:H7. This finding suggests possible changes in culinary practice that may benefit consumers. Another study found that cinnamaldehyde, cinnamon oil, carvacrol, oregano oils, 2,5-dihydroxybenzaldeyde, and 2-hydroxy-2-methoxyenzaldehyde inactivated Mycobacterium avium paratuberculosis that infects cattle and contaminates raw milk. Because there are no effective treatments for this organism, this finding offers new possibilities to protect cattle and milk against this virulent pathogen.